microRNA-34a promotes DNA damage and mitotic catastrophe

Abstract

Efficient and error-free DNA repair is critical for safeguarding genome integrity, yet it is also linked to radio- and chemoresistance of malignant tumors. miR-34a, a potent tumor suppressor, influences a large set of p53-regulated genes and contributes to p53-mediated apoptosis. However, the effects of miR-34a on the processes of DNA damage and repair are not entirely understood. We explored tet-inducible miR-34a-expressing human p53 wild-type and R273H p53 mutant GBM cell lines, and found that miR-34a influences the broad spectrum of 53BP1-mediated DNA damage response. It escalates both post-irradiation and endogenous DNA damage, abrogates radiation-induced G₂/M arrest and drastically increases the number of irradiated cells undergoing mitotic catastrophe. Furthermore, miR-34a downregulates 53BP1 and inhibits its recruitment to the sites of DNA double-strand breaks. We conclude that whereas miR-34a counteracts DNA repair, it also contributes to the p53-independent elimination of distressed cells, thus preventing the rise of genomic instability in tumor cell populations. These properties of miR-34a can potentially be exploited for DNA damage-effecting therapies of malignancies.

Introduction

Competent and error-free DNA repair is crucial for maintaining the integrity of nuclear DNA and preventing chromosomal rearrangements that can lead to genomic instability and cancer.¹ In cancer cells, DNA damage repair can contribute to resistance to radio- and chemotherapy. These modalities are currently the foremost standard treatments for GBM and other malignant brain tumors, which are linked with risk factors, such as exposure to IR and disturbances in the processes of DNA repair, cell cycle regulation, and detoxification.²

DNA damage response is an extremely complex network of sequential processes that coordinate cell cycle progression, DNA repair, and cell death mechanisms.¹ 53BP1 is a remarkable example of DNA repair intricacy. Initially discovered as a regulator of p53-dependent transcription,³ 53BP1 is now considered to be an essential component of the cellular response to genotoxic stress. As a substrate for ATM signaling, 53BP1 relocates together with many proteins into the sites of DNA DSB,⁴ which can be visualized as discrete IRIF. 53BP1, in turn, stimulates ATM activity and plays a role in modulating the chromatin structure around DNA damage sites through direct interaction with methylated histone residues and chromatin-modifying proteins. 53BP1 co-localizes with γH2AX, the well-known DSB surrogate marker, and acts downstream of the plethora of γH2AX-dependent factors that are physically and functionally required for the recruitment of 53BP1 to the sites of DNA DSB.¹ Analogous to its role in immunoglobulin class switch recombination, 53BP1 promotes NHEJ of DSB and fusion of uncapped telomeres by suppressing 5′-end resection⁵ and increasing chromatin mobility.⁶ In response to IR, 53BP1 contributes to p53 accumulation, activation of the intra-S-phase checkpoint, and G₂/M checkpoint arrest.^7,8 The failure of cells to recruit 53BP1 to the sites of DNA DSB results in RIDDLE (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties) syndrome.⁹ In the absence of genotoxic stress, 53BP1 forms nuclear bodies in the sites of spontaneous DNA lesions resulting from the replication stress or during G₂/M transition.¹⁰

MicroRNAs are small non-coding RNA molecules that regulate gene expression. They bind to their mRNAs target sites, resulting in gene silencing through mRNA degradation and/or translational repression. miR-34a, a member of the miR-34 family, is transactivated by the tumor suppressor p53,¹¹ which is frequently mutated in various cancers. The loss of p53 and subsequent inactivation of miR-34a result in activation of oncogenic signaling.¹² miR-34a inhibits cell proliferation, cell cycle progression, cell survival, cell invasion, stemness,^13-15 and epithelial–mesenchymal transitions.¹⁶ miR-34a is downregulated in many cancers, and its levels often correlate with patients survival and prognosis. Deregulation of miR-34a is linked to the resistance of tumors to chemotherapy.^17-21 miR-34a influences a large set of genes that are known to be similarly regulated by p53.²² This supports the idea that by acting downstream and independently of p53, miR-34a can potentially be used as a therapeutic agent for the treatment of both p53 wild-type and p53-mutant tumors. miR-34a contributes to p53-mediated apoptosis and responds to DNA damage in a p53-dependent manner.^23,24 However, the miR-34a-mediated impact on genome integrity is not entirely understood.

Here, we examine the link between miR-34a and DNA damage in human p53 wild-type and R273H p53-mutant GBM cell lines. We find that miR-34a affects 53BP1 expression and translocation to the sites of DNA DSB, leads to an increased propensity of DNA damage, and predisposes irradiated cells to aberrant mitotic progression. We conclude that, by shifting the outcome of the DNA damage response toward MC rather than promoting efficient DNA repair, miR-34a contributes to genome surveillance and prevents the rise of genomic instability in tumor cell populations.

Results

Tet-inducible miR-34a-overexpressing cell lines

To achieve sustained expression of miR-34a, we engineered and selected U87 (p53 wild-type) and U251 (p53 mutant) glioma cell lines expressing high levels of miR-34a upon tet-induction (see “Materials and Methods”, Fig. S1A). Subsequent experiments showed robust production of miR-34a in U87-GFP-miR-34a and U251-GFP-miR-34a cells, which reached its peaks around the 11th day after the beginning of Dox treatment (Fig. S1B). Because of the mutation in p53 DNA-binding domain, a basal miR-34a expression in U251 was not detected. Although U251 induction also resulted in sustained high-level miR-34a expression, its concentration was significantly lower than that in tet-induced U87 line. This could be explained by the rapid elimination of miR-34a-overexpressing cells and overgrow of the initially small (about 1%) but highly proliferative fraction of tet-irresponsive U251 cells. Both U87 and U251 miR-34a-overexpressing cells were characterized by slower proliferation (Fig. S2), increased size, and vacuolization (Fig. S3). The control tet-inducible cells expressing only GFP, as well as regular non-inducible U87 and U251 lines, did not produce extra amounts of miR-34a upon Dox treatment, and their proliferation capacity and morphology were not changed.

miR-34a increases the frequency of chromosome breaks

First, we assessed the effect of miR-34a on the appearance of Mn, small extra-nuclear chromatin-containing bodies that result from unrepaired chromosome breaks or lagging chromosomes. In general, the numbers of Mn significantly vary in undamaged cells, but they rise in response to genotoxic stress and usually correlate with its magnitude. The frequency of spontaneous Mn events was substantially higher in U251- than in U87-derived cell lines (on average, about 2 Mn per cell and 2–5 Mn per 100 cells, respectively), which reflected elevated genomic instability of U251 cells. X-ray irradiation of U251 cells produced even higher numbers of Mn, thus making it difficult to discriminate between IR-induced chromosomal breaks and nuclear fragmentation, which is characteristic of MC (see below). Therefore, we quantified Mn only in U87 cells, used the cytokinesis block (see “Materials and Methods”) to evaluate Mn caused solely by IR, and excluded from the analysis cells with more than 3 Mn. The dependence of Mn frequency in U87 cells on the X-ray dose was determined in preliminary experiments (Fig. S4A). When irradiated at the dose of 4 Gy, U87-miR-34-overexpressing cells demonstrated increasing numbers of Mn per 100 cells with the highest amounts at 11th day after tet induction, when the levels of miR-34a reached their maximum (Fig. 1A). Contrary to miR-34a-overexpressing cells, there was no increase of Mn in control cells at any time point. Without IR, the amounts of Mn remained approximately the same in all cell lines, except for miR-34a-overexpressing cells, in which the number of Mn doubled by 11th day after tet-induction (p < 0.05). Withdrawing Dox from the cultured undamaged miR-34a-overexpressing cells gradually reduced Mn to the amounts comparable with those in control tet-induced and non-induced cells (data not shown). The results suggested that miR-34a counteracted repair processes of generated de novo chromosome breaks and/or enhanced DNA damage both in irradiated and non-irradiated cells.

Figure 1. miR-34a increases DNA damage. See also Figure S1. (A) The numbers of micronuclei (Mn) in miR-34a-overexpressing cells after tet-induction. Black bars indicate miR-34a-overexpressing cells; stars * and **, statistical significance of P < 0.05, and P < 0.005, respectively; X-ray irradiation dose, 4 G. The frequency of spontaneous Mn events was substantially higher in U251- than in U87-derived cell lines (on average, about 2 Mn per cell and 2–5 Mn per 100 cells, respectively), which reflected elevated genomic instability of U251 cells. X-ray irradiation of U251 cells produced even higher numbers of Mn, thus making it difficult to discriminate between IR-induced chromosomal breaks and nuclear fragmentation, which is characteristic of mitotic catastrophe. (B) miR-34a increases the number of spontaneous 53BP1 foci. (1 and 2), U87-GFP-miR-34a non-induced and induced cells respectively; (3 and 4), U251-GFP-miR-34a non-induced and induced cells. Scale bars, 25 µm. (C) Average numbers of spontaneous 53BP1 foci per nucleus. Stars show a statistically significant difference (P < 0.05).

miR-34a overexpression is associated with intensified endogenous DNA damage

In unperturbed cells DNA damage is low compared with that after genotoxic insults. However, 53BP1-positive foci/nuclear bodies, which usually co-localize with many other DNA damage repair factors, have been found in normally proliferating mammalian cells¹⁰ and in preneoplastic and neoplastic tissues in vivo.²⁵ Such “spontaneous” foci represent endogenous DNA lesions resulting from replicative and oxidative stresses, transcription errors, dysfunctional telomeres, and genomic instability of malignant cells.^10,26 In addition, the long-lasting 53BP1 foci, which are suggested to be the sites of incomplete DNA DSB repair, are observed for as long as 14 weeks after the exogenous genotoxic stress.²⁷ 53BP1 is detected in PML nuclear bodies, which are implicated in DNA damage repair,^10,27-29 and in so-called OPT (Oct-1, PTF, transcription) domains, which shield resulting from the replication stress DNA lesions against their degradation to DSB.^10,28 In aging/senescent cells, uncapped telomeres are recognized as DNA DSB and attract many DNA damage response proteins including 53BP1.^29,30

Our results indicated that miR-34a overexpression even in undamaged cells was associated with a higher occurrence of unrepaired chromosomal breaks. To test the possible connection between miR-34a and endogenous DNA damage, we assessed the frequency of spontaneous 53BP1 foci/nuclear bodies in relation to miR-34a levels. 53BP1 foci were present in undamaged normal human lung fibroblasts and human astrocytes but in larger quantity in U87 and U251 GBM cells (AK, and RA, unpublished data), which corroborated the reported prevalence of γH2AX foci in malignant cell lines as compared with primary cell cultures.³¹ Analogous to the higher incidence of Mn events in U251 cells, the number of spontaneous 53BP1 foci in U251 cells exceeded that of U87 cells. miR-34a overexpression caused a shift toward nuclei with higher numbers of 53BP1 foci and augmented the fraction of foci-positive cells, in particular, cells with ≥3 foci (Fig. 1B and C; Table S1).

53BP1-OPT domains were described as large (2–3 µm) discrete nuclear bodies.²⁸ We observed significant variations in size and dispersal of spontaneous 53BP1 foci both in control and miR-34a-overexpressing cells. As reproducible quantification of these small foci was problematic, we took advantage of the software ImageJ (see “Materials and Methods”), which allowed us to detect the tiny spots of higher intensity of fluorescence. The use of the software was validated in preliminary experiments with cells subjected to the low doses of IR (Fig. S4B). Again, after miR-34a overexpression we could see higher numbers of small foci per nucleus, more nuclei with 53BP1 foci, and a larger fraction of cells with ≥3 foci (Table S1). Withdrawal of Dox from miR-34a-overexpressing cells was followed by the gradual reduction of 53BP1 spontaneous foci numbers (data not shown). Tetracyclines have been reported to induce mild DNA damage;^32,33 however, we could not see any disparity between non-induced GFP-miR-34a-cells and control cells expressing only GFP that were treated with Dox for 6 d. Hence, the observed shift toward nuclei with higher numbers of 53BP1 foci was associated exclusively with increased miR-34a levels. Altogether, the prevalence of Mn and increased focal accumulation of 53BP1 suggested escalating endogenous DNA damage events and/or failing DNA damage response in miR-34a-overexpressing but otherwise unperturbed cells.

miR-34a disturbs mitotic progression in irradiated cells

DNA damage and abnormal mitosis, the 2 key promoters of genomic instability, are mutually linked. ³⁴ During mitosis, 53BP1 and a number of γH2AX-dependent factors fail to localize on the condensed chromosomes and to form IRIF.^35,36 The aberrant mitotic progression results in accumulation of de novo DNA damage, which can be propagated to the next cell cycle but usually initiates permanent cell cycle arrest and cell death.^34,37 To explore the impact of miR-34a on mitosis, we examined microscopically asynchronous cells subjected to the low IR doses (1.3 Gy and 1 Gy for U87 and U251 cell lines, respectively) and related mitotic phases to the presence of 53BP1 IRIF. IR at low doses activates the G₂/M checkpoint⁷ and elicits various pathways of DNA repair response.¹ Most of the IRIF in U87 and U251 cells were formed by 40 min following IR, which suggested the onset of G₂/M checkpoint activation. In accord with previous reports,^35,36 the intensely stained 53BP1 appeared to be excluded from the chromatin. However, in irradiated miR-34a-overexpressing cells, 53BP1 foci and aggregates could be visible not only during telophase/cytokinesis,³⁸ but also in prophase. The frequency of mitotic cells was relatively high (about 0.9%) in U251 non-irradiated cells, including control tet-inducible-cells that expressed only GFP, and lower (about 0.3%) in irradiated cells that did not overexpress miR-34a. The highest numbers of mitoses were detected in irradiated U251 miR-34a-overexpressing cells (about 3.5%), and most of the mitotic cells within this group displayed metaphase nuclear patterns (Fig. 2). The frequent presence of mitotic cells could not be attributed to active cell cycling, since proliferation of irradiated cells was slowed within the next 48 h and at least during 1 wk in non-irradiated miR-34a-overexpressing lines (AK, RA, publication in preparation). The results suggested that miR-34a suppressed the G₂/M checkpoint in irradiated cells and delayed mitotic progression before the metaphase–anaphase transition.

Figure 2. miR-34a causes aberrant mitosis. (A) The frequency of mitotic events in irradiated miR-34a-overexpressing cells; the star indicates a statistically significant difference (P < 0.05). (B) 53BP1 foci in U251 mitotic cells at different stages of mitosis. The intensely stained 53BP1 appears to be excluded from the chromatin. Residual 53BP1 foci and aggregates are detectable in prophase (1) and prometaphase (2), absent in metaphase (3 and 4), and re-appear in telophase/cytokinesis (5 and 6); scale bars, 20 µm. 53BP1 is visualized with Alexa^®594, nuclei are counterstained with DAPI.

miR-34a promotes mitotic catastrophe

Impaired G₂/M checkpoint, as well as defects in mitosis, in particular, metaphase arrest, often results in MC. The latter is linked with senescence and considered to be a significant cause of DNA damage-induced cell death.³⁹ We previously found that in vitro and in vivo phenotypes of miR-34a-overexpressing experimental tumors exhibited markers of senescence and MC (AK, RA, publication in preparation). To assess the cumulative effect of miR-34a and IR on triggering MC, we tested various X-ray doses and duration of miR-34a induction. Either IR at 4 Gy or tet-induced miR-34a overexpression for 5 d alone caused MC in about 9% of cells. In contrast, significantly higher numbers of MC cells (35.6% in U87 and 25.8% in U251) could be observed 48 h after IR at 4 Gy, preceded by tet-induced overexpression of miR-34a for 5 d. Both p53wt and p53mut cells responded equally, demonstrating the essential morphological features of MC: gigantism, multiple or multi-branched nuclei, nuclear fragmentation, nuclear blebs, and micronuclei. MC cells often failed to express the GFP reporter and harbored significant amounts of 53BP1 foci, which indicated their completion or premature exit from mitosis, and were strongly positive for the senescence-associated β-galactosidase (Fig. 3). Neither Dox alone nor a combination of Dox and IR in non-inducible cells caused the observed effects. The higher numbers of MC cells in U87 lines as compared with U251 could be explained by p53wt activity, which regulates MC and is implicated in G₁ cell arrest upon acquisition of post-mitotic DNA damage.³⁹ The results indicated that miR-34a promoted MC and cellular senescence in irradiated cells.

Figure 3. miR-34a promotes senescence and mitotic catastrophe (A) Mitotic catastrophe (MC) in irradiated miR-34a overexpressing cells. (1) irradiated U87- and (2) irradiated U251-miR-34a-overexpressing cells demonstrate gigantism, multiple or multi-branched nuclei, nuclear fragmentation, nuclear blebs, and micronuclei. (3 and 4), prevalence of MC events in irradiated induced U87-miR-34a-overexpressing cells (3) vs. non-irradiated U87-miR-34a overexpressing cells (4). (B) The percentage of MC cells in irradiated miR-34a-overexpressing U87 and U251 lines as compared with control groups. White and black columns represent non-irradiated and irradiated cells, correspondingly; * and ** indicate statistical significance of P < 0.05, and P < 0.01, correspondingly; IR dose, 4 Gy. (C) Presence of 53BP1 foci in MC cells indicating completion or exit from mitosis; U87 (1) and U251 (2) irradiated miR-34a-overexpressing cells. Loss of GFP expression in ailing MC cells (indicated by arrows). Overlay of AlexaFluor^®594 (red) and DAPI (blue) (3) and AlexaFluor^®594 and GFP (green) (4). (D) Senescence-associated β-galactosidase in MC cells.

In addition, long-term observation revealed that the cells that survived miR-34a overexpression contained smaller fractions of tet-responsive cells as compared with the cells initially used in the experiments: about 87% survived vs. initial 99% for U87 and about 54% vs. 99% for U251 cells. Consequently, Q-RT-PCR analysis indicated that miR-34a levels were significantly lower in the cells, which survived tet-induction, than in the original lines before prolonged induction (data not shown). The results can be explained by the survival and selection of cells with the lowest levels of tet-inducible miR-34a expression, as well as by the expansion of the initial 1% fraction of tet-irresponsive cells. These data serve as an additional proof that the observed effects are miR-34a-dependent.

miR-34a downregulates 53BP1 expression

In response to IR, 53BP1 contributes to p53 accumulation, activation of the intra-S-phase checkpoint, and G₂/M checkpoint arrest.^7,8 53BP1 has been reported to co-localize on kinetochores together with the centromere-associated protein CENP-E, and to undergo hyperphosphorylation in response to misaligned metaphase chromosomes and damaged spindle microtubules.⁴⁰ 53BP1 interacts with APC component Cdc27 and with Snm1, whose inactivation overcomes mitotic delay and results in the appearance of Mn and supernumerary centrosomes.⁴¹ Both low dose irradiation of 53BP1^−/− cells,⁷ and treatment of normal cells with the radiomimetic drug phleomycin in combination with the Chk-1/Chk-2 inhibitor AZD7762³⁵ have been shown to increase amounts of mitotic cells, presumably due to G₂/M checkpoint release. 53BP1 also acts as a MC suppressor.^42,43 Altogether these data suggested that 53BP1 might be involved in miR-34a-mediated effects related both to DNA damage/repair and mitotic progression.

To address the question of whether 53BP1 is a component of miR-34a signaling pathway, we first tested the effects of IR and adenovirus-mediated p53 (AdP53) overexpression on miR-34a and 53BP1. miR-34a levels were moderately (about 1.5 times) increased in U87 but not in U251 cells upon X-ray irradiation at 2 Gy with no impact on 53BP1 expression. AdP53 rescued miR-34a expression in U373 and LN-Z308 cells, increased miR-34a levels in U87 cells, and moderately downregulated 53BP1 expression at the transcriptional level in all 3 cell lines (Fig. S5A and B). However, the extent of 53BP1 inhibition was insignificant, which could be explained by the delayed upregulation of miR-34a upon AdP53 delivery as compared with transduction with miR-34a-expressing lentiviral vectors (data not shown). In addition, since about 90% of AdP53-targeted cells were undergoing apoptosis by the fourth day after transduction, we could not monitor the effect of p53 on 53BP1 beyond this time point.

We next searched for predicted miR-34a targets in 53BP1 protein-encoding transcripts (53BP1 gene, Ensemble code ENSG00000067369) using several algorithms and databases (see “Experimental Procedures”). While no miR-34a target sites could be found within algorithm-defined 3′UTR boundaries, 8 non-canonical, non-conserved miR-34a binding sites were predicted within the 53BP1 protein-coding region (Fig. S5C) as analyzed by the RNA22 algorithm that allows non-Watson–Crick G–U matches. After being induced for 7 d, miR-34a-overexpressing U87 and U251 cells exhibited lower levels of 53BP1 RNA transcript and protein. In contrast, no changes of 53BP1 expression could be observed in control GFP cells and regular U87 and U251 lines treated with Dox. 53BP1 levels gradually declined due to the prolonged Dox treatment, yet the withdrawal of Dox restored 53BP1 expression in U87 cells (Fig. 4). The results indicated that miR-34a negatively regulated 53BP1 expression in a dose-dependent manner.

Figure 4. miR-34a downregulates 53BP1. See also Figure S2. (A and B) Tet-induced miR-34a overexpression; the seventh day after the beginning of Dox treatment. Red and green cones show the basal and augmented levels of miR-34a; white and black bars indicate the corresponding levels of 53BP1 transcripts as assessed by Q-RT-PCR. (C and D) Removal of Dox from the 7th till the 12th day abrogates miR-34a production and rescues 53BP1 expression. The concentrations of miR34a and 53BP1 transcripts are normalized to the levels of U6snRNA, and to β-actin and hypoxanthine phosphoribosyltransferase-1, respectively; “tet” denotes induced or treated with Dox cells; “reg” stays for unmodified non-inducible U87 and U251 lines; stars indicate a statistically significant difference (P < 0.05).

miR-34a inhibits relocation of 53BP1 to the sites of DNA DSB

The delayed disassembly of 53BP1 IRIF at the beginning of mitosis could reflect the delayed 53BP1 kinetics. We examined whether miR-34a influenced the capacity of 53BP1 to move to the sites of DNA DSB. In non-irradiated cells, 53BP1 was equally distributed within the nuclear space. In control U87 and U251 cells subjected to IR at doses of 1.3 Gy and 1 Gy, respectively, 53BP1 quickly formed discrete IRIF in almost all cells. Preceded by overexpression of miR-34a for 7 d, IR failed to elicit formation of regular IRIF. Instead, the cells demonstrated diffuse staining with irregularly distributed 53BP1 rather than well-distinguished nuclear foci. We calculated average amounts of 53BP1 IRIF in cells 40 min after IR and found them significantly reduced in miR-34a-overexpressing cells as compared with controls (P < 0.05). The fractions of diffusely stained cells in miR-34a-overexpressing cells also exceeded those in the control cells and were inversely correlated with the average numbers of IRIF per cell (Fig. 5). The results showed that miR-34a suppressed the ability of 53BP1 to relocate to the sites of DNA DSB.

Figure 5. miR-34a delays formation of ionizing radiation-induced 53BP1 foci. (A) Gray scale. (1) U87-miR-34a non-induced and (2) U87-miR-34a-tet-induced cells; (3) U251-miR-34a non-induced and (4), U251-miR-34a-tet-induced cells. Scale bars, 50 µm. (B) 53BP1 irradiation-induced foci (IRIF) are visualized with Alexa^®594, nuclei are counterstained with DAPI. Impaired 53BP1 kinetics in irradiated miR-34a-overexpressing cells. (1) U87-miR-34a non-induced and (2) U87-miR-34a-tet-induced cells; (3) U251-miR-34a non-induced and (4) U251-miR-34a-tet-induced cells. Efficient formation of 53BP1 foci in the absence of miR-34a overexpression. (5) U87 non-induced and (6) U87 tet-induced cells; (7) U251 non-induced and (8) U251 tet-induced cells, respectively. (C) Numbers of IRIF per cell, and fractions of cells with diffusely stained 53BP1. Black bars, miR-34a-overexpressing cells; stars indicate a statistically significant difference (P < 0.05); r, Pearson correlation coefficient between diffusely stained cells and IRIF.

Discussion

The reported earlier upregulation of miR-34a in some, although not all, tested cell lines subjected to IR^23,44-46 suggests its participation in DNA damage response. Furthermore, IR-induced p53 phosphorylation at Ser15 and modifications of histones H3 and H4 have been proposed as mechanisms for enhanced miR-34a transcription in irradiated cells.⁴⁷ With rare exceptions, miR-34a is transactivated by p53 wild-type. In GBM cell lines harboring a p53 mutation within the DNA-binding domain, miR-34a expression is abrogated (AK, RA, publication in preparation). Because of its tumor-suppresing capacity, the potential use of miR-34a for the complex therapy of both p53wt and p53mut tumors is widely speculated.⁴⁸ It has also been reported that in Caenorhabditis elegans as well as in breast cancer cells subjected to the high doses of IR (up to 400 Gy) the elevated levels of miR-34a may render radioresistance.⁴⁹ LD₅₀ dose of IR in humans is estimated to be about 4 Gy.⁷ Therefore, we devised our studies within the appropriate dose range and utilized tet-inducible cells lines that provided for the sustained miR-34a expression. This experimental paradigm allowed us to explore a new role of miR-34a as a tumor suppressor, in which miR-34a participates in genome surveillance.

The role of miR-34a in mitotic progression of irradiated cells has been of interest to us for several reasons: (1) we previously found that miR-34a induced morphological changes characteristic for MC even in the absence of genotoxic stress; (2) it is acknowledged that, for G₂/M and mitotic checkpoints to be passed, 53BP1 should be removed from the sites of DNA damage before cells enter mitosis;³⁶ (3) 53BP1 nuclear bodies are cell cycle-regulated, and restoration of DNA or chromatin at the aberrant replication loci requires mitotic transmission;²⁸ ( 4) 53BP1 OPT domains protect lesions generated during G₂/M transition and mitosis;¹⁰ (5) Mn may result from errors in chromosomal segregation during the aberrant mitosis, which is usually accompanied by chromosomal breakage and DNA damage;⁵⁰ (6) 53BP1 is reportedly a positively acting component of the G₂/M checkpoint and APC and a suppressor of MC.^7,8,40-43

Our results indicate that miR-34a increases fragmentation of chromosomes and boosts formation of spontaneous 53BP1 nuclear foci, thus evidencing escalation of ongoing DNA damage, even in the absence of exogenous genotoxic stress. When combined with IR, miR-34a overexpression causes delayed and aberrant progression through mitosis, which is marked by accumulation of cells in metaphase and by the appearance of characteristic for the MC cell morphotypes. In addition, miR-34a downregulates 53BP1 expression and inhibits 53BP1 recruitment to the sites of DNA DSB. We did observe quantitative differences in miR-34a effects when using p53 wild-type and p53 mutant cell lines. It can be explained by p53 wild-type activity and/or some basal levels of miR-34a in non-induced U87 cells as compared with undetectable expression of miR-34a in non-induced p53 mutant U251 cells. However, our data suggest that the described effects of miR-34a are p53-independent. Of note, despite the negative impact of miR-34a on IRIF formation, miR-34a-overexpressing cells display increased amounts of spontaneous 53BP1 foci. This can be interpreted as intensification of endogenous DNA damage resulting from the miR-34a-mediated accumulation of mitotic cells (G₂/M release and/or delay in metaphase) and MC. IRIF are formed quickly and, upon some reports, can be detected as early as 5 min after IR,⁵¹ whereas formation of spontaneous foci takes much longer. This may account for the observed lower amounts of IRIF in miR-34a-overexpressing cells, although not spontaneous 53BP1 foci. Escalation of endogenous DNA damage will add to the burden of DNA damage repair, especially if 53BP1 is downregulated. miR-34-mediated mitotic delay, during which 53BP1 is attached to kinetochores and unable to mark DNA lesions, further highlights the vulnerability of miR-34a-overexpressing cells to exogenous genotoxic insults.

Multiple non-canonical, non-conserved miR-34a target sites with non-Watson–Crick G–U matches within the protein-coding region may be of weaker seed-pairing stability or not as functional as compared with the conserved sites within the 3′UTR.⁵² Nevertheless, our results indicate that miR-34a negatively regulated 53BP1 expression in a dose-dependent manner. Diffuse 53BP1 staining and reduced amounts of IRIF in miR-34a-overexpressing cells, as well as the delayed disappearance of 53BP1 from IRIF at the beginning of mitosis reflect the impaired 53BP1 kinetics and, consequently, the negative impact of miR-34a on DNA repair. While the mechanisms of 53BP1 targeting by miR-34a are yet to be elucidated, we suggest that 53BP1, which activity spreads from the non-homologous end joining to shielding endogenous DNA lesions and preventing aberrant mitotic progression, is a component of miR-34a-signaling pathways (Fig. 6). The increased intensity of endogenous DNA damage after miR-34a overexpression can be attributed to abrogation of the G₂/M checkpoint and/or block of metaphase–anaphase transition. The aberrant mitotic progression is followed by de novo DNA damage, which, due to miR-34a action, will remain unrepaired and unconstrained during the passage through the next G₂/M checkpoint. Hence, miR-34a contributes to DNA damage, yet eliminates the cells with perturbed genomic integrity by shifting the balance from efficient DNA repair to MC. The latter is considered as an oncosuppresive mechanism, which is activated during aberrant mitosis or results from any incorrect division of genetic material.^39,53 Our study reveals a new role of miR-34a as a tumor suppressor, in which miR-34a participates in genome surveillance and prevents the rise of genomic instability in tumor cell populations. These properties of miR-34a can serve as a rationale for its use as a genotoxic sensitizer together with other DNA-damaging agents for the treatment of GBM and other malignant tumors.

Figure 6. A hypothetical model of miR-34a and 53BP1 effects on genomic integrity and mitotic progression. Red-colored elements represent miR-34a and its actions.

Materials and Methods

Cell lines

U87 (ATCC^® #HTB-14^™) and U373 cell lines (discontinued) were obtained from ATCC. U251 (ECACC #09063001) was a kind gift from Dr Benjamin Purow, University of Virginia. LN-Z308 was a kind gift from Dr Erwin Van Meir, Emory University, Atlanta, GA; human astrocytes were a kind gift of Dr Russ Pieper, University of California San Francisco, CA.

Viral vectors and tet-inducible cell lines

miR-34a-GFP- and control GFP-expressing lentiviral vectors were generated by co-transfecting 293T cells with pMIRH34a-PA1 (miR-34a) and pCDH-CMV-MCS-EF1-copGFP (control) plasmids, and the pPACK1™ Lentivector Packaging mix (SBI Biosciences). The titer of the vectors was determined by transduction of 293T cells followed by flow cytometry analysis of the number of GFP-positive cells. To generate miR-34a-tet-inducible cells, the fragment of genomic DNA encompassing miR-34a (NCBI Reference Sequence NR_029610.1) together with its flanking sequences was cloned into pPS-TRE-GFP-miR plasmid (SBI Biosciences) containing copGFP as a gene reporter. The control plasmid was made by inserting the linker instead of miR-34a. The resulting lentiviral vectors expressing miR-34a and/or GFP and Tet-On transactivator pPS-rtTA3R-Puro construct (SBI Biosciences) were used to co-transduce human glioma U87 cells (ATCC #HTB-14) with the following selection for puromycin-resistant tet-induced GFP-positive cells. As pPS-TRE-GFP-miR plasmids are bicistronic constructs, in which both miR-34a and GFP expression are driven by a tet-inducible promoter, we used low concentration (0.05 µg/ml) of doxycyclin hydrochloride (Fisher, Cat.# BP26535) in order to sort out GFP-positive cells that produced the highest levels of miR-34a. The control U87-tet-inducible cells expressed only GFP. Highly responsive U251-GFP-miR-34a-tet and control U251-GFP-tet cells were generated using the same approach. Wild-type p53 and control (empty) adenoviral vectors were a kind gift from Dr Bert Vogelstein, Johns Hopkins University, Baltimore, MD.

Lenti- and adenoviral transduction and tet-induction

To evaluate the effects of miR-34a overexpression, the cells to be tested were grown for 2 d without antibiotics and plated onto 10 cm petri dishes at 100 000 cells/dish. The miR-34a-expressing and control lentiviral vectors were added to the plated cells immediately at the multiplicity of infection of 2 in complete medium (2 ml/dish). Eighteen hours later, 8 ml of fresh medium was added to each dish, and the cells were grown for 5 d. For tet-induced miR-34a overexpression, U87-GFP-miR-34a-tet and control U251-GFP-tet cells were plated at 100 000 cells/dish, and Dox was immediately added at 1 µg/ml. Both lentiviral-transduced and tet-induced cells were collected for quantitative RT-PCR on the fifth day after induction. To evaluate the effects of wild-type p53 overexpression on miR-34a, U373, LN-Z308, and U87 cells were plated onto 10 cm petri dishes at 200 000 cells/dish. Wild-type p53-overexpressing and control adenoviral vectors were added into the culture medium 24 h later at the multiplicity of infection of 1. The cells were collected for quantitative RT-PCR 72 h after transduction.

Flow cytometry

Selection of highly Dox-responsive miR-34a-inducible and control cell lines was performed on the iCyt Reflection Cell Sorter (iCyt Corporation). The cells with the highest levels of GFP fluorescence after treatment with 0.05 µg/ml of Dox (72 h after induction) were sorted out, expanded, and sorted again. The purity of the selected highly responsive to Dox cell population was verified by flow cytometry analysis on the FACSCalibur (BD Biosciences). The cells were subjected to several cycles of expansion, induction, and sorting, until the purity of GFP-positive cells was not less than 99% of the whole population.

Cell proliferation

Cells were seeded onto 10 cm petri dishes at the initial amount of 50 000 cells/dish, split after they reached about 80–90% confluence, collected on the third, fifth, and seventh days, and the ratio between the total and initial amounts was calculated and expressed as “folds”.

Ionizing irradiation

Cells were irradiated by using SARRP device (Xstrahl) using 220 kVp X-ray beam.

Quantitative reverse transcription–real-time PCR

Low molecular weight RNA-enriched fractions were extracted using an Ambion mirVana microRNA Isolation Kit (Life Technology/Applied Biosystems) following the manufacturer’s protocol. Q-RT-PCR was run on 7900HT Sequence Detection System (Applied Biosystems). Quantification of miR-34a levels was performed with Assay OOO426 (Applied Biosystems) using Taqman detection method in accordance with the manufacturer’s instruction (Life Technology/Applied Biosystems). U6 small nuclear RNA (Assay OO1973, Applied Biosystems) was used as a normalization control. The levels of miR-34a were quantified using the ΔΔCt method. Quantitative gene expression analysis of mRNAs was performed with SYBR Green I Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Primers for 53BP1 mRNA matched to 53BP1 transcript versions encoding full-length protein coding frames (1977, 1975, and 1972 amino acids, Ensemble codes ENST00000382044, ENST00000450115, and ENST00000263801, respectively) and were designed using PrimerQuest software http://www.idtdna.com/Scitools/Applications/Primerquest/, Integrated DNA Technologies) as follows: atcagaccaa cagcagaact tcctgg (forward-1) and ttatgggcac ttgaatggtg ctgc (reverse-1), tgtggtgtca caagagtggg tgat (forward-2) and cacaatctcc acgatagcag ggaa (reverse-2). Relative gene expression levels were calculated using the ΔΔCt method with geometric means of the housekeeping genes: Homo sapiens β-actin – gctcctcctg agcgcaagt (forward) and cgtcatactc ctgcttgctg at (reverse), and hypoxanthine phosphoribosyltransferase-1 – tggtcaaggt cgcaagctt (forward) and gggcatatcc tacaacaaac ttgtc (reverse). The results were presented as “fold-difference” in relation to the baseline samples. The changes in the levels of 53BP1 transcripts were construed as a proof of the impact of miR-34a on 53BP1 expression only if they were statistically significant (P < 0.05) and reproduced with 2 independent Q-RT-PCR primer assays.

Western blotting

Evaluation of 53BP1 expression by western blotting was performed according to NuPAGE^® instructions (Invitrogen). Briefly, cells were collected in RIPA buffer with protease inhibitors, mixed with NuPAGE^® LDS Sample Buffer (4×), heated at 70 °C for 10 min, separated on the gel, transferred to nitrocellulose membrane, blocked, probed with 53BP1 antibodies (Cat. #NB100-904, Novus Biologicals) and secondary HRP-labeled antibodies (Cat. #G21234, Invitrogen) and treated with SuperSignal^® West Pico Chemiluminescent Substrate (Cat. #34077, ThermoScientific ). Loading control was performed with antibodies against GAPDH and actin (Cat.# SC-166545 and sc-1616, Santa-Cruz Biotechnology, Inc).

Immunocytochemistry

To visualize 53BP1 nuclear foci, micronuclei, and mitotic progression we utilized 53BP1 antibodies (Cat. #NB100-904, Novus Biologicals) and secondary AlexaFluor^®594-labeled antibodies (Cat. #A11012, Invitrogen). The cytoplasm was stained with AlexaFluor^®488-labeled ConA (Cat. #C11252, Invitrogen), and nuclei were counterstained with DAPI.

Assessment of 53BP1 foci, micronuclei, and aberrant mitoses

Fixed samples were stained with 53BP1 antibodies without nuclear counterstaining by DAPI. Random photos of cells were acquired at magnification ×400 in colored and greyscale formats under identical conditions using the microscope Olympus BX51. The average 53BP1 IRIF number per cell was counted in a minimum of 300 cells, subtracted by the number of spontaneous large 53BP1 foci in mock-irradiated cells, and equilibrated against control samples stained with only secondary antibodies. For the count of spontaneous small 53BP1 foci, the threshold of fluorescence intensity was set up; total 53BP1 foci were calculated with the software ImageJ ; and the number of visually counted large foci was deducted from the number of total foci. The use of the software was validated in the preliminary studies of cells irradiated at low doses when IRIF numbers did not exceed 5 IRIF per cell. The analysis was performed under the same experimental settings.

The formation of Mn was assayed using the cytokinesis block technique. Briefly, cytochalasin B (Cat. #14930-96-2, Sigma-Aldrich) was added into the culture medium at the final concentration 2 µg/ml 6 h after IR. After incubation for 24 h, cells were fixed and stained with DAPI and ConA labeled with AlexaFluor^®488. The number of micronuclei was counted in at least 500 binucleated cells with no more than 3 Mn per cells in at least 20 random fields of view. The results were expressed as the average numbers of Mn events per 100 cells. The cells that had not been treated with ChB were counted as those undergoing MC if they had 3 and more Mn and/or the typical for MC phenotype.

To assess mitotic progression, the G₂/M boundary was detected microscopically upon the nuclear membrane disassembly, characteristic chromatin structure and rounding of the cell prior to cytokinesis. Cells undergoing mitotic catastrophe were characterized by gigantism, multiple or multi-branched nuclei, nuclear fragmentation, nuclear blebs, and multiple micronuclei.

Software

For prediction of miR-34a target sites, the following algorithms and databases were used: miRanda, TargetScan, PITA, RNA22, and MicroCosm. ImageJ was used for the quantitative analysis of acquired images.

Statistics

One-tail distribution, 2-sample unequal (heteroscedastic) variance t test was applied.

Glossary

Abbreviations:

GBM

glioblastoma multiforme

miR-34a

microRNA-34a

53BP1

p53-binding protein-1

IR

ionizing radiation

ATM

Ataxia Telangiectasia Mutated

DSB

double strand breaks

IRIF

ionizing radiation-induced foci

γH2AX

phosphorylated histone 2AX

NHEJ

non-homologous end joining

APC

anaphase-promoting complex

MC

mitotic catastrophe

Mn

micronuclei

Dox

doxycyclin

PML

promyelocytic leukemia

10.4161/cc.26510

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/cc/article/26510