Programmed expression of pro-apoptotic BMCC1 during apoptosis, triggered by DNA damage in neuroblastoma cells
- 218 Downloads
The multi-functional BMCC1 (BCH motif-containing molecule at the carboxyl terminal region 1)/PRUNE2 plays a clear role in suppression of tumor activity. In the patients with neuroblastoma (NB), reduced expression of BMCC1 in primary tumor tissues was associated with poor prognosis. By contrast, enforced expression of BMCC1 as well as elevated expression of BMCC1 in response to DNA-damage promotes apoptosis by abrogating Akt-mediated survival pathways.
We addressed molecular mechanisms underlying changes in regulation of BMCC1 expression during the process of apoptosis, which was promoted by a DNA-damaging drug Cisplatin (CDDP), in NB-derived cells.
Elevated expression of BMCC1 was identified as an early response to DNA damage, which is accompanied by phosphorylation of ataxia telangiectasia mutated kinase (ATM) and accumulation of E2F1. Indeed, inhibition of ATM using an ATM inhibitor resulted in a decrease in expression of BMCC1 at mRNA levels. In addition, an E2F-binding sight was required for activation of BMCC1 promoter in response to DNA damage. On the other hand, knockdown of E2F1 yielded abrogated induction of BMCC1 in the cells after treatment with CDDP, suggesting that BMCC1 accumulation was caused by ATM-E2F1-dependent transcription. Finally, we demonstrated that full-length BMCC1 was proteolytically cleaved by apoptosis-activated caspase-9 during advanced stages of apoptosis in SK-N-AS cells.
In this study, we demonstrated the programmed expression of full-length BMCC1 in human NB cells undergoing DNA damage-induced apoptosis. The elucidation of the molecular mechanisms controlling the regulation of BMCC1 during apoptosis initiated by DNA damage provides useful information for understanding drug resistance of tumor cells and spontaneous regression of NB.
KeywordsBMCC1 E2F1 Apoptosis Caspase-9 DNA damage Neuroblastoma
Ataxia telangiectasia mutated
ATM and RAD3-related
BNIP2 and Cdc42GAP homology
BCL2-homology domain 3
BNIP2 and Cdc42GAP homology motif-containing molecule at the carboxyl terminal region 1
Fetal bovine serum
Forkhead box O3a
Mre11, Rad51 and Nbs1
Nerve growth factor
Poly [ADP-ribose] polymerase 1
Reverse transcription polymerase chain reaction
Neuroblastoma (NB) is a common malignant solid tumor in children, which originates from the sympathoadrenal lineage of neural crest. When NB is diagnosed at age less than one year, it can regress spontaneously (even for the metastatic stage), resulting in a favorable prognosis . However, details of the molecular mechanism responsible for this spontaneous regression remain obscure .
Human BMCC1 (BCH motif-containing molecule at the carboxyl terminal region 1)/PRUNE2 encodes a 340-kDa protein with a conserved BNIP-2 and Cdc42GAP homology (BCH) scaffold domain on its C-terminus [3, 4, 5]. Previous studies have demonstrated that BCH domain can modulate signaling networks and affect multiple cellular functions, such as morphogenesis, differentiation, motility, and apoptosis . Therefore, functional contributions of BMCC1 in the regulation of signaling networks and multiple cellular functions, including apoptosis, have been suggested.
Our previous research has demonstrated that BMCC1 promotes neuronal apoptosis caused by nerve growth factor (NGF)-depletion . In addition, BMCC1 can initiate and promote apoptosis via its C-terminal BNIP-2 homology region by inhibiting multiple steps in Akt-mediated survival pathway, as observed in NB and non-NB cells . Increased expression of BMCC1 in response to DNA damage has linked to phosphorylation of ataxia telangiectasia mutated (ATM) at Ser-1981, corresponding to its sensitivity to DNA-damaging drugs . Recently, we found that BMCC1 was trans-activated by E2F1 during normal cell cycle by binding to BMCC1 promoter in human cells . However, precise expression profile of BMCC1 and effects on the apoptotic process initiated by DNA damage remain unclear.
E2F transcription factors can modulate expression of a series of genes associated with cell cycle progression, apoptosis, and DNA repair . Among the E2F family proteins, molecular contribution of E2F1 in the progression of apoptosis caused by DNA damage has been characterized. In apoptotic cells triggered by DNA damage, E2F1 accumulates by stabilization of protein levels and not by transcriptional induction of mRNA levels [8, 9, 10, 11, 12]. Stabilization and subsequent activation of E2F1 following DNA damage can be partially modulated by DNA damage-phosphorylated ATM/ATM, RAD3-related (ATR), and Chk2 [12, 13, 14], which results in elevated expression of pro-apoptotic transcription targets, such as p73 [15, 16, 17] and Apaf1 .
DNA damage facilitates accumulation of intrinsic apoptosis [19, 20, 21], which promotes breakdown of mitochondrial membrane and cytosolic release of cytochrome C. Then it can bind to Apaf1 to induce activation of caspase-9 through proteolytic cleavage . Subsequently, downstream caspases, such as caspase-3, caspase-6, and caspase-7, are activated through their proteolytic cleavage by active caspase-9 . Similarly, the BNIP family proteins, which include BNIP-2 and BNIP-XL, are also cleaved by in vitro activated caspases, although their functional contribution to apoptotic process was not elucidated .
In this study, regulatory mechanism of BMCC1 in apoptosis of human cells initiated by Cisplatin (CDDP) was investigated. Elevated expression of full-length BMCC1 associated with early responses to DNA damage and reduced expression of BMCC1 in apoptotic cells were found. A further analysis demonstrated that acute induction of BMCC1 at transcriptional levels was mediated by E2F1. Subsequent reduction of BMCC1 during apoptosis was due to the proteolytic cleavage by caspase-9. The findings of this study provide evidence for a better understanding of the tumor suppressive function of BMCC1, as a signal for apoptosis of cells with DNA damage.
Materials and methods
Cell culture, treatments, cell viability, knockdown and overexpression
Human NB-derived SK-N-AS, NBL-S, and NLF cells were obtained from the Children’s Hospital of Philadelphia cell line bank and were maintained in RPMI 1640 culture medium (Wako, Osaka, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) and 100 U/mL Penicillin-Streptomycin (Thermo Fisher Scientific). LNCaP cells were provided from Dr. Ueda [5, 25] and were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated FBS and 100 U/mL Penicillin-Streptomycin. The cells were grown in 5% CO2 and humid condition at 37 °C . Cisplatin (CDDP, Sigma-Aldrich, St. Louis, MO) was used to induce DNA damage, and 2-Morpholin-a-yl-6-thianthren-1-yl-pyran-4-one (ATM kinase inhibitor, Calbiochem, San Diego, CA) was used to inhibit ATM activity. To inhibit the activity of caspases, SK-N-AS cells were treated with 40 μM of CDDP and were cultured in the presence of 50 μM of a pan-caspase inhibitor (Z-VAD-FMK, Promega, Madison, WI) or a caspase-9-specific inhibitor (Z-LEHD-FMK, R&D systems, Minneapolis, MN). After 48 h of CDDP treatment, cell viability was measured using WST-8 assay kit (Dojindo, Kumamoto, Japan), and yielded absorbance on 450 nM was detected with Microplate Reader MTP-310 (HITACHI, Tokyo, Japan). For knockdown of E2F1 using lentiviral shRNA delivery system, MISSION shRNA plasmid DNA clones (shE2F1#1 (TRC 0000000251) and shE2F1#2 (TRC 0000000252)) were purchased from Sigma-Aldrich.
Semiquantitative RT-PCR and quantitative real-time PCR
Total RNA was extracted from NB cell lines using RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was reverse-transcribed from total RNA using SuperScript II reverse transcriptase and random primers (Thermo Fisher Scientific) and subjected to PCR-based amplification using rTaq DNA polymerase (TaKaRa, Shiga, Japan). The primer sets used for PCR were as follows:
BMCC1 forward: 5′- GAAGCCTCTGGTCCAGTCAG-3′;
BMCC1 reverse: 5′-CTTCGGCCGTATATTCTGGA-3′;
E2F1 forward: 5′-TGCAGAGCAGATGGTTATGG-3′;
E2F1 reverse: 5′-GTTCTTGCTCCAGGCTGAGT-3′;
TAp73 forward: 5′-CCTCTGGAGCTCTCTGGAAC-3′;
TAp73 reverse: 5′-GAAGACGTCCATGCTGGAAT-3′;
GAPDH forward: 5′-ACCACAGTCCATGCCATCAC-3′;
GAPDH reverse: 5′-TCCACCACCCTGTTGCTGTA-3′.
A GeneAmp PCR 9700 and a Veriti thermal cycler (Thermo Fisher Scientific) were used for PCR, and the amplified DNA fragments were separated using agarose gel electrophoresis. Then, they were stained with ethidium bromide and detected with a UV-transilluminator (ATTO, Tokyo, Japan). The expression levels of BMCC1 in SK-N-AS cells were measured by quantitative real-time PCR (Q-PCR) using ABI PRISM 7500 system (Thermo Fisher Scientific). TaqMan probes for BMCC1 (Hs00322421_m1) and GAPDH (4310884E) were purchased from Thermo Fisher Scientific.
Whole cell lysate were immunoblotted as previously described . The primary antibodies used for immunoblotting were described as follows. The rabbit antibody against human BMCC1 was generated by MBL (Nagoya, Japan) . The Anti-E2F1 (#3742), anti-phospho-ATM (Ser1981) (#4526), anti-phospho-Chk2 (Thr68) (#2661), anti-phospho-H2A.X (Ser139) (#9718), anti-phospho-FOXO3a (Thr32) (#9464), anti-FOXO3a (#2497), anti-PARP (#9542, Fig. 5a and b) and anti-caspase-9 (#9502), anti-Bim (#4582), HRP-conjugated anti-Rabbit (#7074) and anti-Mouse (#7076) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-Human phospho-E2F1 (pS364) was purchased from ROCKLAND (Limerick, PA) and anti-p73 (ab-3) was obtained from Millipore (Billerica, MA), whereas anti-ATM (sc-23,921), anti-PARP-1 (sc-8007), and anti-Actin (sc-8432) were bought from Santa Cruz Biotechnology (Dallas, TX).
Luciferase reporter assay
Luciferase reporter plasmids harbored human BMCC1 promoter region from − 1727 to + 78 (pGL3-BMCC1-luc promoter luciferase vector), a deletion mutant of BMCC1 promoter (pGL3-BMCC1-luc △S1/S7 (△ − 1727/− 532)), and two point mutants of putative E2F1 binding constructs (pGL3-BMCC1-luc-△S1/S7 + S8 mut (− 329/− 322) and pGL3-BMCC1-luc-△S1/S7 + S9 mut (− 9/− 2)) (Fig. 3a). The luciferase reporter assay follows a previously described protocol .
In vitro caspase cleavage assay
Proteins were synthesized from the T7 promoter on pcDNA6.2-BMCC1 or pcDNA3.1-MDM2 plasmids using in vitro transcription and translation (TNT) kit (Promega). To label this protein with biotin, TNT reaction was performed in the presence of biotinylated lysine-tRNA (Promega). Biotinylated proteins reacted with recombinant active caspase-9 (Millipore, Billerica, MA) or active caspase-3 (Millipore), supplied for SDS-PAGE, transferred onto a PVDF membrane, and detected using HRP-conjugated Streptavidin. Developed signals were captured using a LAS-4000 imager.
All quantitative experiments were replicated at least three times independently. Error bars indicated standard deviation (SD). Statistical analysis was done by student t-test and p-value was calculated using Excel software (v14.7.3) (Microsoft, Redmond, WA).
Expression profile of BMCC1 in apoptotic SK-N-AS cells induced by CDDP treatment
Acute induction of BMCC1 was associated closely with accumulation of E2F1 in cells with DNA damage
E2F1 promoted apoptosis, triggered by DNA-damaging agents, was mediated via accumulation through protein stabilization, not by transcriptional induction [8, 9, 10, 11, 12]. In addition to stabilization of E2F1, phosphorylation of E2F1 at Ser-364, catalyzed by Chk2 after phosphorylation by ATM/ATR, was associated with its transcriptional activity . Indeed, up-regulation of E2F1 protein levels (and not mRNA levels) was detected in SK-N-AS cells shortly after CDDP treatment (Fig. 2a and b). Furthermore, phosphorylation of E2F1 at Ser-364 catalyzed by p-Chk2 was observed in these cells along with increased amounts of p-Chk2 (Fig. 2a and b). Again, when ATM-dependent DNA damage signal pathway was blocked in NB cells with an ATM inhibitor, there was a reduction in the immediate induction of BMCC1 both at mRNA and protein levels, normally followed by accumulation of E2F1 in response to DNA damage (Fig. 2b, c, Additional file 3: Figures S3a and b). On the other hand, ATM inhibitor influenced marginal effect on E2F1 mRNA expressions in NB cells with DNA damage (Fig. 2c and Additional file 3: Figure S3b). These findings suggest that transcriptional induction of BMCC1 mediated by DNA damage is closely associated with ATM-dependent stabilization of E2F1.
ATM activity and an E2F-binding sight was required for activation of BMCC1 promoter in response to DNA damage
CDDP treatment enhanced the luciferase activity driven by BMCC1 promoter, but the enhancement was completely blocked in the presence of the ATM inhibitor (Fig. 3b). These results imply that CDDP-dependent activation of BMCC1 promoter is strongly associated with ATM activity. In addition, by using deletion and mutant constructs of BMCC1 promoter (ΔS1/S7, ΔS1/S7&S8-mut, and ΔS1/S7&S9-mut, Fig. 3a), it was demonstrated that the effect of CDDP treatment on BMCC1 promoter depends on site-9 (Fig. 3c). This result was consistent with our previous finding that E2F binding site-9 was essential for BMCC1 promoter activity . Therefore, these results indicate that transcriptional induction of BMCC1 mediated by DNA damage is regulated by E2F1-binding site S9 in its promoter region.
E2F1 was essential for induction of BMCC1 in response to DNA damage
Next, we wondered whether acute induction of BMCC1 triggered by DNA damage is dependent on E2F1. To this end, we conducted shRNA-mediated knockdowns of E2F1 in SK-N-AS cells, which were exposed to DNA damaging drugs.
We further monitored DNA damage using elevated amounts of p-ATM, p-Chk2, and p-H2A.X. A significant attenuation of DNA damage caused by CDDP in E2F1-knockdown SK-N-AS cells was found (Fig. 4a). Because the accumulation of p-H2A.X is a marker for DNA double-strand breaks, the results suggest that knockdown of E2F1 can attenuate the cellular response toward DNA double-strand breaks.
Down-regulation of full-length BMCC1 in apoptotic cells through a caspase-9-dependent mechanism
A previous in vitro study found that variants of BMCC1, known as BNIP family (e.g., BNIP-2 and BNIP-XL), could be cleaved efficiently by several caspases . Therefore, it was assumed that full-length BMCC1 can also be cleaved by caspases during apoptosis. The potential amino acid sequences within full-length BMCC1 protein cleaved by caspase activity were searched, and two caspase-9 cleavage sites, LEID (660–663) and LEED (2380-2383), were identified (Additional file 4: Figure S4). The active caspase-9 can cleave proteins with LEXD sequence [27, 28], and thus the cleavage of full-length BMCC1 by caspase-9 in the apoptotic cells was verified. A pan-caspase inhibitor (z-VAD-FMK) and caspase-9-specific inhibitor (z-LEHD-FMK) were used to inhibit caspase activation during apoptosis initiated by CDDP. SK-N-AS cells were treated with 40 μM of CDDP and cultured in the presence or absence of caspase inhibitors for 24 h (Fig. 5b). Immunoblotting results revealed that pan-caspase and caspase-9-specific inhibitors efficiently block the accumulation of cleaved forms of caspase-9 and PARP-1 in SK-N-AS cells treated with CDDP. At the same time, we found that the reduced expression of BMCC1 in the cells treated with CDDP was significantly reversed by inhibition of caspase-9. These observations indicate that reduced expression of full-length BMCC1 within apoptotic cells was strongly associated with caspase-9 activation.
Proteolytic cleavage of full-length BMCC1 by caspase-9
Finally, we investigate whether caspase-9 could directly cleave full-length BMCC1. To this end, biotin-labeled full-length BMCC1 was synthesized using TNT system  and subjected to in vitro caspase proteolysis assay (Fig. 5c). As expected, the full-length BMCC1 (340 kDa) was reduced efficiently in a dose-dependent manner following incubation with a recombinant active caspase-9. The smaller fragments of 70, 60, and 45 kDa bands were observed as a result of the treatment with active caspase-9. In addition, the recombinant active caspase-3 could not cleave full-length BMCC1, while it cleaves its target MDM2 . Therefore, it was affirmed that the full-length BMCC1 is proteolytically cleaved by caspase-9 during apoptosis.
Recently, we reported that acute expression of BMCC1 during apoptosis is initiated by DNA-damaging drugs, and is required for progression of apoptosis in human cells . We also reported that BMCC1 is transcriptionally up-regulated via transcription factor E2F1-dependent mechanism involving E2F binding site S9 in the cell cycle of NB cells . Here, it was noticed that transcriptional induction of BMCC1 during apoptosis is regulated by E2F1-dependent mechanism via E2F binding site S9, in association with accumulation of E2F1 after CDDP treatment. Furthermore, requirement of ATM activation for the E2F1-dependent transcriptional up-regulation of BMCC1 in response to DNA damage in SK-N-AS cells was proven by inhibition of DNA damage response initiated by CDDP using an ATM inhibitor. As previously reported, overexpression of BMCC1 in human cells induces apoptosis by inhibiting multiple steps in Akt-survival signal and by DNA-damaging drugs . Therefore, similar to pro-apoptotic function of p73 in cells with DNA damage [15, 16, 17], BMCC1 also functions in apoptosis initiated by DNA damage through elevated expression by E2F1. Hence, these findings point out that BMCC1 expression induced by an E2F1-dependent mechanism may act in sensitizing cells to DNA damage and eliminating cells with DNA damage(s) by promoting apoptosis.
In addition to E2F1 role in apoptosis of DNA-damaging cells, which is modulated downstream of ATM/ATR activation [8, 9, 10, 11, 12, 13], E2F1 is involved in promoting DNA repair through phosphorylation of p53 and Chk2 by an ATM- and Nbs1-dependent mechanism [30, 31]. This is accompanied by the use of proteins involved in DNA repair, such as Mre11, Rad51, and Nbs1 (MRN) complex, at sites of DNA double-strand breaks [32, 33]. By contrary, we observed that knockdown of E2F1 in SK-N-AS cells led to attenuated levels of p-ATM and p-H2A.X, two DNA double-strand break indicators in the early stages of apoptosis. Similarly, knockdown of E2F1 in SK-N-AS cells inhibited the expression of BMCC1 in normal culture conditions , preventing even under DNA-damaging conditions.
In the later stages of apoptosis, reduced amounts of full-length BMCC1 were accompanied by cleavage of caspase-9 and PARP-1. Furthermore, in vitro experiments demonstrated that full-length BMCC1 was proteolytically cleaved by a recombinant active form of caspase-9. Therefore, these observations suggest that reduced expression of full-length BMCC1 in apoptotic cells was directly regulated by activated caspase-9. Although full-length BMCC1 can promote apoptosis through BCL2 binding and Akt inhibition with its BNIP-2 homology C-terminus , the fragment(s) of BMCC1 cleaved by caspase-9 may also acquire new functions during apoptosis progression. Thus, in well agreement with previously reported results on BH3-containg pro-apoptotic proteins, such as Bid , Bad  and Bim , which enhance apoptotic cascade, a pro-apoptotic role of caspase-cleaved fragments with BH3 domain of BNIP family proteins, including BMCC1, was identified in this study.
From a clinical point of view, BMCC1 is considered as a tumor suppressor. BMCC1 is expressed widely in normal cells, but its expression is decreased in skin, prostate, and colon cancers . Attenuated expression of BMCC1 is associated with aggressive NB, and thus it is a prognostic indicator of NB . In addition, BMCC1 has a clear effect in suppressing the prostate cancer . Furthermore, loss of functional mutations in the coding region of BMCC1 has been identified in merkel cell carcinoma  and parathyroid carcinoma , but the functional role of these mutations in promoting apoptosis initiated by DNA damage remains to be elucidated.
The present study uncovered the programmed expression (E2F1-dependent transcriptional induction and caspase-9-dependent proteomic cleavage) of full-length BMCC1 in the course of apoptosis triggered by DNA damage. Our current findings may provide greater insight into tumor suppressive functions, such as sensitivity to DNA-damaging agents and spontaneous regression of BMCC1 in NB and other cancers.
We thank Ms. Natue Kitabayashi, Ms. Hiromi Isano and Mr. Tomoaki Yokoyama for their excellent technical assistance. We also thank Dr. Takeshi Ueda for providing LNCaP cells. We are grateful to Drs. Toshinori Ozaki, Osamu Shimozato, Hiroki Nagase and Masatoshi Tagawa for their critical reading of the article, fruitful discussions and suggestions.
MSI, RT, TY, AN, and YT conceived and designed the experiments. MSI, RT, JA, and YT performed the experiments. MSI, RT, YN, AN, and YT contributed to prepare reagents and experimental materials. MSI, RT, TY, and YT wrote the paper. All authors read and approved the final version of the manuscript.
This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare for Third Term Comprehensive Control Research for Cancer, JSPS KAKENHI (Grant Numbers 17 K10132, 26461602, 22791507 and 24249061), the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct) from Japan Agency for Medical Research and Development (AMED), the National Cancer Centre Research and Development Fund (4), and a Grant from Takeda Science Foundation. The funding bodies had no involvement in the design of the study and collection, analysis, and interpretation of data and in writing this manuscript.
Ethics approval and consent to participate
Ethics approval is not required in this study.
Consent for publication
The authors declare that there are no competing financial interests in relation to the work described.
- 3.Machida T, Fujita T, Ooo ML, Ohira M, Isogai E, Mihara M, Hirato J, Tomotsune D, Hirata T, Fujimori M, et al. Increased expression of proapoptotic BMCC1, a novel gene with the BNIP2 and Cdc42GAP homology (BCH) domain, is associated with favorable prognosis in human neuroblastomas. Oncogene. 2006;25(13):1931–42.CrossRefGoogle Scholar
- 27.Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP, et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. functional relationships established for key mediators of apoptosis. J Biol Chem. 1997;272(29):17907–11.CrossRefGoogle Scholar
- 38.Yu W, McPherson JR, Stevenson M, van Eijk R, Heng HL, Newey P, Gan A, Ruano D, Huang D, Poon SL, et al. Whole-exome sequencing studies of parathyroid carcinomas reveal novel PRUNE2 mutations, distinctive mutational spectra related to APOBEC-catalyzed DNA mutagenesis and mutational enrichment in kinases associated with cell migration and invasion. J Clin Endocrinol Metab. 2015;100(2):E360–4.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.