Human LINE-1 retrotransposon induces DNA damage and apoptosis in cancer cells

Abstract

Background: Long interspersed nuclear elements (LINEs), Alu and endogenous retroviruses (ERVs) make up some 45% of human DNA. LINE-1 also called L1, is the most common family of non-LTR retrotransposons in the human genome and comprises about 17% of the genome. L1 elements require the integration into chromosomal target sites using L1-encoded endonuclease which creates staggering DNA breaks allowing the newly transposed L1 copies to integrate into the genome. L1 expression and retrotransposition in cancer cells might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome instability. There is however little evidence on the mechanism of L1-induced genetic instability and its impact on cancer cell growth and proliferation.

Results: We report that L1 has genome-destabilizing effects indicated by an accumulation of gamma-H2AX foci, an early response to DNA strand breaks, in association with an abnormal cell cycle progression through a G2/M accumulation and an induction of apoptosis in breast cancer cells. In addition, we found that adjuvant L1 activation may lead to supra-additive killing when combined with radiation by enhancing the radiation lethality through induction of apoptosis that we have detected through Bax activation.

Conclusion: L1 retrotransposition is sensed as a DNA damaging event through the creation DNA breaks involving L1-encoded endonuclease. The apparent synergistic interaction between L1 activation and radiation can further be utilized for targeted induction of cancer cell death. Thus, the role of retrotransoposons in general, and of L1 in particular, in DNA damage and repair assumes larger significance both for the understanding of mutagenicity and, potentially, for the control of cell proliferation and apoptosis.

Figure 1

Experimental strategy for assaying RC-L1 expression and retrotransposition. (A) L1 element consists of the 5'- and 3'-UTRs and two ORFs. The EGFP retrotransposition cassette is cloned into the L1 3'-UTR in the antisense orientation. L1 elements tagged with the EGFP cassette are cloned into the pCEP4-based mammalian expression vector with puromycin resistance gene. RC-L1-EGFP-expressing cells are sorted by FACS and retrotransposition is detected by EGFP fluorescence using fluorescence microscopy. (B) Confirmation of L1 retrotransposition by PCR as revealed by a 342 bp product. MCF-7 cells-expressing L1-EGFP. Serial passaged cells (P1–P4) show the spliced form of EGFP at 342 bp. No DNA template used as negative control. L1-EGFP plasmid showing the unspliced form at 1243 bp used as a positive control. 1 Kb ladder used as a molecular weight marker.

Figure 2

RC-L1 is expressed in breast cancer cells. RC-L1- expression determined by immunofluorescence using anti-L1 ORF1 (A) and anti-L1 ORF2 (B) rabbit polyclonal antibodies showed cytoplasmic "C" and nucleolar "N" staining. No specific staining was detected when using pre-immune serums as control. Immunoblotting of whole cell lysates using anti-ORF1 (C) or anti-ORF2 (D) detected a 40 kD and a 150 kDa respectively. Additional bands were detected by anti-ORF2 antibody at 135 kDa and 66 kDa which may be due to cleavage by a cellular protease. P1 through P4 correspond to consecutive passages of RC-L1 expressing cells. Anti-α-tubulin was used as a loading control. Mock transfected cells serve as a negative control.

Figure 3

Cell cycle and DNA damage response analysis of RC-L1-expressing cells. MCF-7 cells either Mock transfected, expressing GFP or RC-L1 were analyzed by FACS as described under "Experimental Procedures". The histograms represent the distribution of cells through the cell cycle measured by flow cytometry and analyzed with ModFit. (A) untransfected MCF-7 cells. (B) MCF-7 cells transfected with EGFP (C) MCF-7 cells transfected with RC-L1. The percentage of cells in G₂ or M is shown for each treatment group. Detection of the induction of γ-H2AX foci formation using anti-γ-H2AX polyclonal antibody. (D) by immunostaining of Mock transfected cells and serially passaged RC-L1-expressing cells (P1-P4) (E) Number of γ-H2AX foci in four different cell passages, P1-P4. Error bars show s.d. (F). Expression level of γ-H2AX determined by immunoblotting.

Figure 4

Induction of apoptosis in breast cancer cells harboring RC-L1. (A) Pro-apoptotic bax gene expression level determined by immunoblotting using anti-Bax polyclonal antibody. Anti-α-tubulin was used as a loading control. Mock transfected cells serve as a negative control. (B). Induction of apoptosis in RC-L1-expressing cells determined using caspase 3 assay.

Figure 5

Impact of RC-L1 expression and retrotransposition on p53 mutant cells. (A) Confirmation of L1 retrotransposition by PCR as revealed by a 342 bp product. (B) The histograms represent the distribution of cells through the cell cycle measured by flow cytometry and analyzed with ModFit. "A" untransfected T47D cells. "B" T47D cells transfected with EGFP "C" T47D cells transfected with RC-L1. (C) RC-L1- expression determined by immunofluorescence using anti-L1 ORF1 and anti-L1 ORF2 rabbit polyclonal antibodies (upper panels). Detection of the induction of γ-H2AX nuclear foci formation using anti-γ-H2AX polyclonal antibody (lower panels). (D) Pro-apoptotic bax gene expression level determined by immunoblotting using anti-Bax polyclonal antibody in T47D cells either expressing RC-L1, or treated with 5Gy radiation or both and assessed at the indicated time points.