HPV-18 E2 protein downregulates antisense noncoding mitochondrial RNA-2, delaying replicative senescence of human keratinocytes

Results

HFK-E2 cells express the HPV-18 E2 oncoprotein and downregulate ASncmtRNAs

To establish whether the E2 protein alone induces downregulation of the ASncmtRNAs in an HPV-negative context, we transduced HFK with lentiviral constructs encoding the green fluorescent protein ZsGreen alone (HFK-ZsG) or ZsGreen and HPV-18 E2 (HFK-E2) and purified cells by sorting, obtaining on the average transduced cell populations of 92% and 78%, respectively (Fig. 1A). Only those cells transduced with HFK-E2 expressed E2 mRNA (Fig. 1B). Immunofluorescence confirmed that only HFK-E2 cells express the E2 protein, localized to the cytoplasm (Fig.1C). Moreover, at population doubling or passage 3 (p3), only HFK-E2 cells showed downregulation of both ASncmtRNAs (Fig. 1D, E, F), while the expression of SncmtRNA was unaffected (Fig. 1D, G).

Figure 1. E2-expressing HFK downregulate the ASncmtRNAs. HFK were transduced in triplicate for 72 h with HPV-18 E2 (HFK-E2) or with control lentivirus (HFK-ZsG) or mock-transduced. (A) Representative analysis of HFK-ZsG and HFK-E2 populations displayed 92% and 78% transduction, respectively. (B) Only HFK-E2 cells expressed the full-length E2 mRNA (1,100 bp). (C) Only HPV-18 E2-transduced HFK expressed E2 protein as evaluated by immunofluorescence (Bars = 40 µm). (D) Relative levels of ncmtRNAs were determined at p3 by RT-PCR using 18S rRNA as loading control. Numbers on the right denote amplicon size in bp. Triplicate analysis of ASncmtRNA-1 (E), ASncmtRNA-2 (F) and SncmtRNA (G) showed that both ASncmtRNAs were downregulated by E2 expression (*p<0.01), while SncmtRNA levels remained unchanged.

E2 oncoprotein extends replicative lifespan of HFK

To evaluate whether HPV-18 E2 is able to induce HFK immortalization, population doubling of transduced cells was measured at different passages. A triplicate determination showed that HFK-E2 cells maintained proliferative activity until becoming arrested at p26 (Fig. 2A). At this stage, expression of E2 was downregulated, as determined by fluorescent immunocytochemistry, compared to HFK transduced with the complete genome of HPV-18 (18Nco cells), used as positive control for E2 expression [14] (Fig 2B). Western blot carried out in triplicate confirmed the progressive downregulation of E2 (Fig. 2C, D). At p3 after E2 transduction, cells strongly downregulated ASncmtRNA-1 and this low expression level remained constant until replicative arrest at p26 (Fig. 2E, F). In contrast, ASncmtRNA-2 also decreased at p3 but gradually increased from p15 up until p26 arrest, where its expression was recovered to the levels found in early passage HFK-E2 cells (Fig. 2E, G). Interestingly also, at p26 HFK-E2 cells downregulated the expression of SncmtRNA (Fig. 2E, H), supporting the hypothesis that this transcript is involved in cell cycle progression [11,12,14,15].

Figure 2. HPV-18 E2 expression extends replicative lifespan of HFK and modulates expression of ncmtRNAs. (A) A triplicate determination of proliferation rate was plotted as time (days) versus cell doubling or passage number (p). Cells were arrested at p26. (B) At p26 the expression of HPV-18 E2 was suppressed as compared to 18Nco cells, transduced with the complete genome of HPV-18 (Bars = 50 µm). (C) E2 protein expression was progressively reduced in HFK-E2, from p8 to p26. Calnexin (CANX) was used as loading control. (D) A triplicate analysis of the results in (C) show a quantification of the gradual decrease of E2 from p8 to p26 (**p=0.001; ***p=0.0001). (E) Determination of the relative expression of ncmtRNAs by RT-PCR using 18S rRNA as loading control. A triplicate analysis shows downregulation of ASncmtRNA-1 at p3 (**p<0.01), which is sustained until p26 (F). In contrast, ASncmtRNA-2 is also downregulated at p3 but is later progressively upregulated from p15 to senescence at p26 (**p=0.001) (G). (H) SncmtRNA is downregulated at p26 (**p=0.001).

E2-expressing HFK arrest at G₂/M and become senescent

In striking contrast to E2-expressing HFKs (Fig. 2), wild-type and ZsGreen control HFKs exhibit an early replicative arrest. A triplicate analysis of population doublings of HFK and HFK-ZsG showed that these cells arrested at p8 (Fig. 3A, B). A representative cell cycle analysis revealed that HFK and HFK-ZsG at p8 were arrested at G₁ (Fig. 3C). These cells arrested at G₁ showed an increase in the activity of the replicative senescence marker SA-β-galactosidase [20] (Fig. 3D, E) together with upregulation of p21 (Fig. 3F, G), as compared to p3 HFK and comparable to HFK cells treated with H₂O₂ as positive control of senescence [21] (see Methods). Analogously to p26-arrested HFK-E2 cells, RT-PCR showed that the relative expression of SncmtRNA was strongly downregulated in p8 HFK (Fig. 4A, B), while, conversely, ASncmtRNA-2 was significantly upregulated (Fig. 4A, C) and ASncmtRNA-1 remained constant (Fig. 4A, D).

Figure 3. HFK and HFK-ZsG undergo senescent arrest in G₁ at p8. (A, B) A triplicate analysis of proliferation rate of HFK and HFK-ZsG was plotted as time (days) versus cell doubling or passage number. Neither cell population proliferated past p8. (C) Flow cytometry of HFK at p1 and p8 and HFK-ZsG at p8 shows that non-E2-expressing cells arrest in G₁ at p8. (D) HFK at p8 were positive for SA-β-galactosidase staining, which was negative in p3 cells. H₂O₂ was used as positive senescence control (Bars = 100 μm). (E) A triplicate analysis of the results shown in D revealed a 4 to 5-fold increase in SA-β-galactosidase activity, compared to p3 cells, and similar to the level of HFK treated with H₂O₂ (***p<0.005). (F) In HFK at p8, p21 was upregulated compared to p3 cells. (G) A triplicate analysis of the experiment in F revealed a 4 to 5-fold increase in p21 levels compared to p3 cells, and at a similar level to H₂O₂-treated cells (***p<0.005).

Figure 4. Changes in ncmtRNA levels in senescent HFK arrested at p8. The ncmtRNAs were amplified by RT-PCR from total RNA purified from HFK at p1, p3, p5 and p8, using 18S rRNA as an internal control. The experiments were run in triplicate and the relative band density was normalized to 18S for each case. (A) Representative gels showing RT-PCR amplification of these transcripts. (B) The relative expression of SncmtRNA was downregulated at p8. (C) In contrast, ASncmtRNA-2 increased progressively from p1 to senescence at p8 (***p<0.005). (D) Relative expression of ASncmtRNA-1 remained constant.

In contrast to wild type HFK and HFK-ZsG, cell cycle analysis of HFK-E2 cells showed normal cell cycle distribution of G₁, S and G₂/M phases at p8, p15 and p20 with a slight tendency of an increase in the proportion of cells at G₀/G₁ (Fig. 5A). However, at p26, cells were arrested at G₂/M, similar to HFK treated with colchicine, used as positive control (see Methods) (Fig. 5A). At p26, HFK-E2 cells became senescent, as observed by SA-β-galactosidase staining (Fig. 5B). Of note, p8 HFK and HFK-ZsGreen cells were elongated and contained a single nucleus (Fig. 5B, upper left). In contrast, HFK-E2 were negative for SA-β-galactosidase at p8 and p16 but positive at p26 (Fig. 5B). Interestingly, p26 HFK-E2 were binucleated, in agreement with G₂/M arrest (Fig. 5B, lower right; see arrowheads at higher magnification).

Figure 5. Spontaneous downregulation of E2 in HPV-18 E2-transduced cells induces replicative senescence. (A) Flow cytometric analysis of HFK-E2 cells at passages 8, 15 and 20 exhibited normal cell cycles with similar proportion of cells at G₀/G₁, S and G₂/M phases. At p26, cells were arrested at G₂/M, similar to HFK treated with 100 nM colchicine as positive control. (B) As shown before, HFK and HFK-ZsG at passage 8 were positive to SA-β-galactosidase activity, whereas HFK-E2 cells at p8 and p16 were negative to this senescence marker (DAPI stain is shown for both fields). At p26, HFK-E2 cells became positive for SA-β-galactosidase staining and a higher magnification shows many binucleated cells (arrowheads). Bars = 50 μm.

Expression of p21 in HFK-E2 cells at p26 was inhibited as compared to p3 HFK or p3 HFK treated with H₂O₂ (Fig. 6A, B). Expression of p21 is dependent on p53 [22–24], and therefore we analyzed whether the expression of this tumor suppressor was also modified. As shown in Fig. 6A and C, the expression of p53 was also downregulated in p26 HFK-E2, compared to p3 HFK. Similarly, the expression of CDK1 (Fig. 6A, D), but not cyclin B1 (Fig. 6A, E), was also downregulated. Notice that treatment of p3 HFK with H₂O₂ inhibited the expression of both CDK1 and cyclin B1 (Fig. 6A, D, E).

Figure 6. HFK-E2 cells at p26 inhibit the expression of p21. (A) Western blot of p21, p53, CDK1 and cyclin B1 in HFK at p3 and HFK-E2 at p26, using H₂O₂ as positive control of senescence. (B) A triplicate Western blot analysis confirms downregulation of p21 in HFK-E2 cells at p26 as compared to p3 HFK (**p<0.01). (C) The p53 protein, which is related to p21 expression, is also downregulated (**p<0.01), similar to CDK1 (D) (**p<0.01). (E) Cyclin B1 expression was not affected in HFK-E2 cells arrested at G₂/M (ns). Of note, expression of both CDK1 and cyclin B1 is downregulated by treatment with H₂O₂. (**p<0.01).

Discussion

Here, we demonstrate that expression of HPV-18 E2 protein induces in HFK downregulation of the ASncmtRNAs, establishing a new function for this viral protein during HPV-18-induced cellular transformation. These results confirm our previous suggestion that the E2 protein might be involved in downregulation of the ASncmtRNAs in cells transduced with the complete genome of HPV-18 [14]. However, this model was complicated to reach a definitive conclusion since these cells also express other HPV proteins. Previously, we reported that in different human cancer cell lines and in 17 human cancer biopsies of different origin including uterine cervix carcinoma, the ASncmtRNAs were downregulated and we proposed that these mitochondrial transcripts behave as tumor suppressors [12]. In a challenging review, Bellanger et al. discussed the possibility of pleiotropic characteristic of HPV E2 functioning as either tumor suppressor or oncogene [5]. The present results confirm that the HPV E2 protein behaves as a tumor suppressor at least in cervical cancer and that downregulation of the ASncmtRNAs constitutes an important step in carcinogenesis [12,15–17,25]. However, which cellular factor(s) induces downregulation of the ASncmtRNAs in non-HPV induced cancers remains unclear. Interestingly however, transfection of primary cultured mouse hepatocytes with antigen X of Hepatitis B virus [26] also induces downregulation of the ASncmtRNAs (E. Jeldes et al., unpublished results), suggesting that proteins encoded in other oncogenic viruses may have similar functions.

Previously, we reported that the SncmtRNA and the ASncmtRNAs are synthesized in mitochondria based on the following results: 1) these transcripts exhibit 99% plus identity with the sequence of the human 16S mitochondrial gene. 2) They are detected in isolated mitochondria treated with ribonuclease A to eliminate transcripts attached to the outer membrane. 3) Ethidium bromide and rhodamine 6G, two drugs that interfere with mitochondrial transcription and mtDNA replication, inhibit the expression of these transcripts [11,12]. Taken together, the effect of E2 on the inhibition of the expression of ASncmtRNAs suggests that this viral factor interacts with the organelle. Indeed, it was reported that HPV-18 E2 localizes to the mitochondrial inner membrane, affects cristae morphology and increases mitochondrial ROS production without inducing apoptosis [27]. This report suggests that E2 might affect the expression of the ASncmtRNAs.

Transduction of HFK with HPV-18 E2 extends the replicative lifespan of HFK up to p20-p23 and later, at p26, cells are arrested and become senescent, concomitantly with downregulation of E2 expression. SncmtRNA becomes downregulated upon senescence, both in HFK and HFK-E2 cells, confirming the role of this transcript in cell proliferation, as previously demonstrated with other cell lines [11–15]. On the other hand, ASncmtRNA-2 is gradually upregulated during progression towards senescent arrest, both in wild-type HFK and E2-transduced cells. ASncmtRNA-1, in contrast, does not suffer the same fate; both in HFK and E2-HFK, the expression level of this transcript remains constant, even after reaching replicative senescence. These results suggest that another characteristic of replicative senescence is upregulation of ASncmtRNA-2 and downregulation of the SncmtRNA. Supporting this conclusion is a previous report on human umbilical vein endothelial cells (HUVEC) that become senescent and arrest at G₂/M after 15 passages. Senescent HUVEC cells express SA-β-galactosidase and p21 and the ASncmtRNA-2 is upregulated [28]. Unfortunately, the expression of SncmtRNA and ASncmtRNA-1 was not evaluated in this report. The differential expression of ASncmtRNA-1 and ASncmtRNA-2 suggests that these two transcripts might play different functions in the cell. Indeed, treatment of the human melanoma cell line SK-MEL-2 with doxorubicin, a drug that induces DNA double-strand breaks [29], upregulates only the expression of ASncmtRNA-1 suggesting that this transcript may be involved in DNA repair (V. Burzio et al. unpublished results).

As described before, senescent HUVEC cells at p15 upregulate the ASncmtRNA-2, together with the expression of hsa-miR-1973 and hsa-miR-4485 [28]. Although in this work we did not determine the presence of these microRNAs, we studied their expression in a different system. We found that knockdown of the ASncmtRNAs in the breast cancer cell line MDA-MB-231 induces a strong increase in the expression of hsa-miR-1973, hsa-miR-4485-3p and hsa-miR-4485-5p, whose sequences are perfectly contained within the inverted repeat of ASncmtRNA-2. An hsa-miR-4485-3p mimic induces a drastic inhibition of cyclin B1 and cyclin D1 (Fitzpatrick et al, submitted).

HFK at p8 and HFK-E2 at p26 display proliferative arrest and exhibit elongated morphology and expression of SA-β-galactosidase, a marker of senescence. However, cell cycle analysis shows a major difference between arrested HFK at p8 and HFK-E2 cells at p26: p8 HFK were arrested at G₁ while at p26 HFK-E2 cells were arrested at G₂/M. In addition, arrested HFK cells contain a single nucleus while HFK-E2 cells are binucleated. More important is the notorious difference in the expression of p21, which is upregulated in p8 HFK and downregulated in p26 HFK-E2. Senescence has been described as an irreversible arrest of cells at G₁ phase [30–33]. However, it is becoming increasingly clear that senescence also takes place at G₂/M [34–37]. The p21 protein plays an important function as inhibitor of the cyclin-dependent kinases CDK1, CDK2 and CDK4/6 and also as an essential mediator of senescence of cells arrested at G₁ and G₂/M [38–42]. The low expression of p21 in HFK-E2 might be explained by downregulation of p53, a tumor suppressor involved in the expression of p21 [39–41]. In addition, we show that CDK1, an essential G₂/M checkpoint factor of the cell cycle [42,43], is also downregulated, further supporting the G₂ arrest. Surprisingly, the expression of cyclin B1, activator of CDK1, is not affected by senescence of HFK-E2 cells. A schematic summary of these results is presented in Fig. 7. These observations might be the result of E2-induced cell cycle deregulation and genome instability [44,45]. Interestingly, Hela, HT-3 and CaSki cells infected with a SV40 vector expressing the bovine papillomavirus E2 protein showed downregulation of p53 and p21, besides other proteins involved in cell cycle regulation [46]. However, these results were obtained with previously transformed cells, contrary to the present work where we used normal untransformed keratinocytes as targets for HPV infection.

Figure 7. Lentiviral transduction of HPV-18 E2 induces downregulation of both ASncmtRNA-1 and ASncmtRNA-2 in HFK. In consequence, replicative lifespan is extended from 8 to 26 passages, when cells become senescent. HFK, at passage 8 (p8), arrest at G₁, concomitant with increase of the p21 tumor suppressor protein. In contrast, E2-expressing HFK arrest at p26 in G₂/M, while p21 decreases and cells are binucleated. Remarkably, in both cases, ASncmtRNA-2 is upregulated and SncmtRNA is downregulated upon senescence, while ASncmtRNA-1 remains downregulated in HFK-E2 and constant in HFK and HFK-ZsG.

In conclusion, these results allow us to define cellular phenotypes according to their proliferative status. Previously, we defined three cellular phenotypes according to the expression pattern of the SncmtRNA and the ASncmtRNAs [11–15]. Non-proliferating cells or cells at G₀ such as human naïve lymphocytes express a trace amount of these transcripts. In contrast, normal proliferating cells express SncmtRNA and the ASncmtRNAs [12]. Remarkably however, tumor cells express the SncmtRNA and downregulate the ASncmtRNAs [12–17]. Here we report that HFK and HFK-E2 reach senescent arrest, together with upregulation of ASncmtRNA-2 and downregulation of the SncmtRNA. Upregulation of the ASncmtRNA-2 was also reported in senescent HUVEC cells arrested at G₂/M [28]. Therefore, a fourth cellular phenotype is represented by senescent cells, in which the expression of SncmtRNA is downregulated while the ASncmtRNA-2 is upregulated.

Acknowledgments

We thank Dr. Luisa L. Villa, Universidade de São Paulo, Brazil, for the kind donation of HPV-transformed 18Nco cells and Dr. Françoise Thierry, Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), Singapore, for the kind donation of a sample of E2 antibody. We also thank Dr. Pablo D.T. Valenzuela for his continuous support.

Conflicts of Interest

L.O.B. holds ownership of equity of Andes Biotechnologies. All other authors declare no potential conflict of interest.

Funding

This work was supported by the “Comisión Nacional de Investigación Científica y Tecnológica (CONICYT), Chile [grants Fondecyt-1140345, Fondecyt-11090060, Fondecyt-1085210, Fondef-D04I1338, AFB 170004 and CCTE-PFB16].

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