Retrotransposition-Competent Human LINE-1 Induces Apoptosis in Cancer Cells With Intact p53

Cell culture, antibodies, and plasmids

HCT-116, SW480, and DLD-1 were maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Gibco-BRL, Calif) with 10% fetal bovine serum (FBS), 1% L-Glutamine, and 1% penicillin-streptomycin, in the presence of 5% CO₂/95% air at 37^°C. HT1080wt, HT1080mut, and MCF-7 were maintained in Iscove's modified Dulbecco's medium (Gibco-BRL) with 10% FBS, 1% L-Glutamine, and 1% penicillin-streptomycin. Normal human fibroblasts (NHF) were maintained in Dulbecco's modified Eagle's medium (DMEM)/H-21 with 10% FBS, 1% L-Glutamine, and 1% penicillin-streptomycin.

The antibodies anti-p53, anti-Bcl₂, anti-Bax, and anti-HRP-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Calif. Anti-α-tubulin was purchased from Sigma (Saint Louis, Mo). The plasmid pJM101 containing human L1 tagged with an antisense NEO cassette was obtained from John V. Moran (University of Michigan Medical School, Mich). The control plasmid, pBRV1 containing only NEO gene under SV40 promoter, was a gift from Vladimir Larionov (NCI, NIH) and has been previously described [24]. The plasmids p1321 (E6), normal E6/E7 and a control E6/E7 deficient plasmid, p1318 (ΔE6) were obtained from Peter M. Howley (Harvard Medical School Mass). The HPV16 E6/E7 containing construct used, p1321, has been described previously [25]. To construct the plasmid, a SalI/HindIII fragment containing the E6/E7 ORFs of HPV16 was ligated to the HindIII/SalI site in the p1318 vector, which contained a human β-actin enhancer/promoter and a β-actin 3′ UTR polyadenylation signal sequence. A control E6/E7-deficient plasmid p1318 was produced by removing the SalI/HindIII fragment.

Cell transfection and L1 retrotransposition assay

5 × 10⁵ cells were seeded in 60 mm dishes and grown to 70% confluence. Transfections were carried out using lipofectamine (Gibco-BRL). 2 μg of plasmid pJM101 DNA and 8 μL of lipofectamine reagent were used for each transfection. pJM101 contains a complete human L1 and is marked with two selectable genes, hygromycin and neomycin [26]. 16 hours after transfection the medium was changed, and 3 days later, Hyg^R cells were selected by growth in the medium containing 250 μg/mL hygromycin for 14 days (Figure 1). Hyg^R cells were trypsinized and plated in growth medium containing 300–400 μg/mL G418. After 14 days the resistant cells (G418^R) were either fixed and stained to score the retrotransposition frequency or collected for DNA and protein extractions.

Retrotransposition frequency and colony forming ability

Retrotransposition frequency was scored as the number of G418^R colonies per 10⁶ HYG^R cells plated. 10⁶ Hyg^R cells were plated in triplicate in the presence of G418 and maintained for 14 days under this selection. Growing clones were fixed, stained with Giemsa stain solution, and counted.

For the colony forming ability, one thousand cells (10³) from HCT-116 and SW480 cell lines, either mock transfected or transfected with L1 and selected for either hygromycin or G418 resistance, were plated in triplicate. Colony forming ability is determined as the number of colonies generated from 10³ plated cells and given as a percentage.

Polymerase chain reaction

Genomic DNA was extracted using DNAzol reagent (Molecular Research Center, Inc, Ohio). PCR was carried out in 50 μL reaction volume. Each PCR reaction contained 4 mM MgCl₂, 5 μL 10 X buffer, 0.2 mM dNTPs, 200 ng of each primer, 10 U of Taq polymerase, and 200 ng of DNA template. The reactions were carried out under the following conditions: 94^°C for 10 minutes, 30 cycles of 94^°C for 1 minute, 64^°C for 30 seconds, and 72^°C for 30 seconds, then 72^°C for 10 minutes. One fifth of the reaction volume was loaded and separated on 1% agarose gels containing ethidium bromide.

Immunoblotting

Cells, either mock-transfected or transfected with pJM101 or pBRV1 (as control), were lysed in the following buffer: 120 mM NaCl, 25 mM Tris, pH 7.5, 1% Triton x-100, and protease inhibitors cocktail (Complete Mini, Roche, Germany). The cells were centrifuged and the proteins collected and quantified using the Bio-Rad assay (Bio-Rad, Calif). The proteins were electrophoresed through 12% SDS-polyacrylamide gels, and transferred to nylon membranes (Immobilon-P). The membranes were first incubated in 5% blocking solution (nonfat dry milk in 1 X PBS) for two hours at room temperature, then incubated in blocking solution overnight at 4^°C with a primary antibody: anti-p53 (1 : 500 dilution), anti-Bcl₂ (1 : 500 dilution), anti-Bax (Santa Cruz Biotechnology) (1 : 250 dilution), or anti-α-tubulin (Sigma, Saint Louis, Mo) (1 : 1000 dilution) as a loading control. The membranes were washed first for 15 minutes and then 4 times for 5 minutes each in PBST (PBS 1 X + 0.1% Tween 20). The blots were incubated in blocking solution for 45 minutes at room temperature with the secondary HRP-conjugated antibody (Santa Cruz Biotechnology), and washed in PBST as described above. The detection was performed using the Western Blotting Luminol Reagent (Santa Cruz Biotechnology).

Apoptosis detection assay

Apoptosis was assayed using TiterTACS kit (Trevigen, Md) following the manufacturer's recommendations on cells plated and fixed in 96-well plates. Cells were either mock-transfected or transfected with L1 plasmid (pJM101) or with NEO plasmid (pBRV1) and selected for G418 resistance. As a control, cells were treated with nuclease allowing complete degradation of DNA. TiterTACS is a colorimetric assay to quantify apoptosis by detecting DNA fragmentation in cells.

Inactivation of p53 by human papillomavirus E6/E7 genes

HCT116 cells were seeded in 6-well dishes (2 × 10⁵ cells/well) and grown to 70% confluence in DMEM-complete media. First, cells were transfected with pJM101 and selected for hygromycin resistance as described earlier. After 14 days, the Hyg^R cells were trypsinized, pooled, and expanded in DMEM-Hyg until 70% confluence. Hyg^R cells were then transfected with either no DNA (mock), 1 μg of complete E6 (E6), or 1 μg of ΔE6 (E6-deleted form) plasmid DNA and 8 μL of lipofectamine reagent. Three days later, the cells were placed in DMEM-complete containing 300–400 μg/mL G418 (DMEM-G418). After 10–14 days, the G418^R cells were fixed and stained with 0.4% Giemsa solution for visualization and counting. The number of G418^R colonies was scored and the retrotransposition frequency was determined.

Human L1 retrotransposition in cancer cells

In order to investigate the impact of human L1 transposition on cancer cell growth, we first established stable transformants using a retrotransposition assay which employed a selectable molecular marker in cultured cells (Figure 1). The assay is based on the excision of an inverted intron from a neomycin phosphotransferase (NEO) gene [26]. Before retrotransposition the intron prevents NEO expression. During retrotransposition the intron is spliced out of the RNA intermediate and allows NEO expression upon reinsertion of the L1. We analyzed two colorectal cancer cell lines with different p53 status: HCT-116 (p53wt) and SW480 (p53mut) for their ability to allow L1 retrotransposition by measuring their colony forming ability before and after transfection with L1 plasmid (Figure 2a). Both cell lines showed similar survivability, and both seem to equally support transfection with L1 and selection with hygromycin, as demonstrated in the colony formation assay. Interestingly, we found a dramatic difference between the two cell lines in the outcome of G418 selection, which allows for selection of cells harboring retrotransposition events. Indeed, while we were able to recover-large number of G418^R clones from SW480 with mutant p53, only a few clones were recovered from HCT-116 cell line with wild-type p53 although the same number (10⁶ cells) of Hyg^R cells were plated for both cell lines to carry out G418 selection (Figure 2a). We concluded that it is the direct effect of retrotransposition in HCT-116 that results in reduced colony survival.

To test whether the G418^R clones generated after transfection have acquired resistance because the tagged L1 elements have undergone retrotransposition, we performed a PCR analysis of these clones using primers flanking the neomycin cassette. The results show that the retrotransposition marker, the neomycin phosphotransferase gene, is correctly spliced as evidenced by the detection of a 468 bp DNA fragment (Figure 2b). This was also shown for HCT-116 and SW480 (Figure 2b, lane 3). As controls, we used genomic DNA from mock-transfected cells, no DNA, or the DNA plasmid alone. Hyg^R cells did not show the spliced form in either cell line (lane 2). In most cases, only the spliced form (468 bp) was visualized on agarose gels, which indicates that only the spliced and active form of neomycin gene can be stably maintained in cells. However, the longer unspliced form (1368 bp) can also sometimes be seen as extrachromosomal molecules in early passage.

To test whether this association between low number of G418^R clones and the p53 status is applicable to other cell lines, we decided to analyze a panel of cell lines from different tissues having either a wild-type or mutant p53. This group included two fibrosarcoma cell lines, HT1080wt (p53 wild type) and its derivative HT1080mut (p53 mutant); a breast adenocarcinoma cell line, MCF-7 (p53wt); and an NHF (p53wt). The retrotransposition frequency was scored as described. As before, cell lines harboring wild-type p53 were poorly supportive of retrotransposition (Table 1). In contrast, the cell line with mutant p53 exhibited higher frequencies of retrotransposition. At this stage, low-frequency retrotransposition can be explained either by the inability of cells expressing p53wt to allow high L1 retrotransposition, or the inability to detect clones undergoing retrotransposition in cells because of their immediate loss.

L1 induction of apoptosis in p53 (wt) cell lines

Since induction of apoptosis is an early event in DNA damage-mediated p53-dependent response and since p53 wild-type cells exhibit a dramatic decrease in their survivability after L1 retrotransposition, we examined the role of apoptosis in the observed differences in retrotransposition frequency. We assayed for apoptosis using a colorimetric assay allowing quantification by detection of DNA fragmentation. A dramatic increase in apoptosis was detected in G418^R HCT-116 cells, while no induction of apoptosis was detected in G418^R SW480 cells (Figure 3a). A nuclease treatment was used as a positive control. To confirm that drug selection using G418 is not associated with the induction of apoptosis, we transfected these cells with a plasmid containing only the NEO gene (pBRV1) without the L1 element. No apoptosis was induced in these cells. Thus, these data demonstrate that the induction of apoptosis is associated with the presence of retrotransposition-competent L1.

To further test whether the induction of apoptosis is directly associated with the presence of L1, proteins extracted from HCT-116 transfected either with the L1 plasmid or with a neomycin plasmid (vector alone) were analyzed by immunoblotting (Figure 3b). The induction of Bax expression was detected only in G418^R cells derived from cells transfected with L1 plasmid (lane 3). No induction of Bax expression was observed in mock-transfected cells (lane 1), Hyg^R cells transfected with L1 (lane 2), or G418^R cells transfected with a plasmid containing only NEO gene (vector alone) (lane 4).

Suppression of p53 by expression of E6 ablates L1-dependent apoptosis

The E6 protein encoded by the oncogenic human papillomaviruses (HPVs) targets p53 for ubiquitin-dependent proteolysis [27]. To address the role of a competent p53 in the induction of apoptosis, we examined the effects of L1 activity in cells in which p53 is functionally inactivated by HPV E6 expression. We used the ability of the HPV E6 gene to inactivate p53 in order to examine the dependence of L1-mediated apoptosis on a functional p53 protein. When HCT-116 cells are transformed with L1 they undergo apoptosis and inhibition of cell growth. When these same cells were transfected to express a normal E6 gene (L1 + E6), the effect of L1 on cell growth is reduced and the number of G418 resistant colonies increased (Figure 4). When a parallel population of HCT-116 cells were transfected to express L1 and a deleted form of E6 (L1 + ΔE6), the L1 retrotransposition frequency was reduced and colony formation was similar to L1 alone. These results are consistent with the notion that L1-induced apoptosis requires a functional p53.

L1 retrotransposition in isogenic HT1080 cells

We have conducted most of our comparative studies in two colorectal cancer cell lines HCT-116 and SW480. These cells differ not only by their p53 status, but also by the status of some DNA repair genes, mainly mismatch repair genes hMLH1 and hMH6. Therefore, we cannot rule out the implication of other genes in the observed differences in these cells ability to undergo apoptosis after the addition of retrotransposition-competent L1 elements. Therefore, we further studied the association between L1 retrotransposition and p53 status in the isogenic cells HT1080wt, with wild-type p53, and its derivative HT1080mut, with mutant p53. HT1080 mutant cells (HT1080TG) have two independent mutations in each of the p53 alleles and can make p53 [28]. We showed that G418^R clones have indeed undergone the retrotransposition for the marked L1 elements, using PCR analysis. The spliced form of 468 bp was detected in both HT1080wt and HT1080mut after transfection and selection for G418 resistance (Figure 5a). We analyzed protein expression levels in HT1080wt and its derivative HT1080mut by immunoblotting before and after transformation with human L1 (Figure 5b). We found that p53 is overexpressed after transfection and selection for G418^R clones harboring retrotransposition-competent L1 elements in both HT1080wt and HT1080mut. We also found that Bax is overexpressed in HT1080wt in a much more pronounced manner in comparison with HT1080mut. We quantified the differences in Bax expression between HT1080wt and HT1080mut by densitometric analysis (Figure 5c). While we found that Bax expression was induced relatively about 8 times in HT1080wt, the difference was less than double in HT1080mut cells. In addition, we observed a decrease in Bcl₂ expression level following L1 retrotransposition in HT1080wt cells (Figure 5d) and to a slightly lower extent in HT1080mut cells (Figure 5e). This is in agreement with p53 being involved in the induction of apoptosis following L1 retrotransposition, and in agreement with our finding in other cell lines where we have observed a more pronounced induction of apoptosis in cells with wild-type p53.

Human L1 as a potential novel cancer therapeutic strategy based on the induction of apoptosis: perspectives

Both chemotherapeutic agents and irradiation can induce tumor cell death primarily by causing DNA damage. However, it has been very difficult to limit damage to normal tissue to an acceptable level using these therapies. Although a number of methods to induce target cells to undergo apoptosis exist and are being evaluated, any approach which can be selectively targeted to the cancer cell offers an advantage to existing methods. The activation of L1 retrotransposition induces genomic instability, thereby triggering a DNA damage response, which results in the delay of cell cycle progression and/or induction of apoptosis. Using an endogenous component of the genome for a preferential induction of apoptosis within tumor cells could provide a novel mode of attack for cancer therapeutics. The employment of sophisticated cell-specific vectors as gene therapy tools will enable targeting of L1 only to tumor cells.