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ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation
Cancer Letters 373 (2016) 214–221
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation Xiao-Juan Ma, Li Shang, Wei-Min Zhang, Ming-Rong Wang, Qi-Min Zhan * State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China
A R T I C L E
I N F O
Article history: Received 30 October 2015 Received in revised form 10 January 2016 Accepted 11 January 2016 Keywords: Genomic stability Nlp UV radiation NER Tumorigenesis
A B S T R A C T
Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030– 1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. © 2016 Elsevier Ireland Ltd. All rights reserved.
Introduction Ultraviolet (UV) irradiation has been linked to skin cancers, including non-melanoma and melanoma skin cancers [1–3]. The major DNA lesions induced by UVB or by high-energy artificial UVC irradiation are cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts; (6-4) PPs]. Usually, CPDs are formed quite significantly much more than (6-4) PPs [4,5]. CPDs formation and repair are important in connection with carcinogenesis in human skin [6]. Nucleotide excision repair (NER) is the main biological event responsible for the removal of bulky, helix-distorting DNA lesions, induced by UV irradiation, environmental mutagens, and certain chemo-therapeutic agents [7,8]. The NER pathway consists of two sub-pathways of NER, global genome NER (GG-NER) and transcription-coupled NER (TC-NER), differing in damage recognition [8,9]. Both sub-pathways require the core NER factors, of which XPA is the central component as it interacts with TFIIH, RPA, XPCRAD23B, DDB2, ERCC1-XPF, and PCNA proteins [7,10–16]. ERCC1XPF is recruited to NER complexes by interaction with XPA to trigger the dual incision reaction [10,17].
* Corresponding author. Tel.: +86 10 67762694; fax: +86 10 67715058. E-mail address: [email protected] (Q.-M. Zhan). http://dx.doi.org/10.1016/j.canlet.2016.01.020 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.
Nlp (ninein-like protein) has been characterized as a centrosomal protein that is essential for proper mitotic events. Nlp is tethered to the centrosome by BRCA1 and is involved in microtubule organization in interphase cells by recruiting γ-TuRCs and displaced from maturing centrosome at G2/M transition to ensure mitotic spindle formation [18–22]. Its subcellular localization and protein stability are regulated by several crucial mitotic kinases, such as Plk1, Nek2, Cdc2 and Aurora B [18,20,23–26]. Deregulation of Nlp in cells results in aberrant spindle, chromosomal missegregation and multinucleus, abnormal cytokinesis and induces chromosomal instability and renders cell tumorigenesis [18,20,23,24,26]. Our previous studies have identified that Nlp interacts with BRCA1 through its C terminus, which is required for normal microtubule nucleation at interphase [22]. Since BRCA1 also functions in DNA damage response [27], we propose that Nlp might implicate in the process of DNA repair. In this work, we show that Nlp plays a role in NER after UV-induced DNA damage. More interestingly, downregulated expression of Nlp is observed in human skin carcinomas. Materials and methods H1299, A549 and HeLa cell lines were purchased from ATCC (American Type Culture Collection). They were cultured under desired medium. For UV treatment, exponentially growing cells or the cells after transfection were rinsed with phosphatebuffered saline (PBS) and irradiated with UV radiation at different doses. After UV irradiation, fresh medium was added and the cells were cultured until harvest. Detailed information is shown in the Supplementary materials.
X.-J. Ma et al./Cancer Letters 373 (2016) 214–221
Results Nlp enhances DNA repair following UV-induced DNA damage In order to determine whether Nlp is involved in DNA damage response, we treated H1299 cells (lung cancer cell line) with IR or UV with appropiate doses. There was little change of subcellular localization of Nlp in the case of IR which mainly caused double strand breaks (DSBs) (data not shown). However, after UV radiation, the localization of Nlp changed obviously (Fig. 1A). Then we asked whether Nlp contributes to photoproducts excision. Host cell reactivation (HCR) assay and the removal of CPDs assay by ELISA were employed. Upon knockdown of Nlp expression in H1299 cell line (Fig. 1B), the HCR activity and the rate of CPDs removal were significantly attenuated compared with the control groups (Fig. 1C and D). Similarly, both the HCR activity and the removal of CPDs were more powerful in HeLa cells (cervical cancer cell line) reconstituted with pEGFP-C3-Nlp and KYSE30 cells (esophageal squamous cell carcinoma cell line) transfected with pcDNA3.1(+)-Nlp than their counterparts (Fig. 1E–G and Supplementary Fig. S4A–C). Consistently, when the removal of CPDs was measured in MEFs derived
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from Nlp transgenic [28] and wild type mice, similiar results were obtained (Fig. 1H). Collectively, the findings indicate that Nlp is involved in the DNA repair process and probably regulates nucleotide excision repair (NER) pathway following UV-radiation. Nlp protects cells from UV radiation-induced cell death Given that Nlp promotes NER activity, we next explored its physiological functions. H1299 cells transfected with Nlp siRNAs, HeLa and KYSE30 cells overexpressing Nlp were treated by UVC radiation with the indicated doses (0 J/m2, 5 J/m2, 10 J/m2, 20 J/m2, 40 J/ m2). Cell survival was then measured at 24 hours (Fig. 2A and B and Supplementary Fig. S4D) or 42 hours (Supplementary Fig. S1A and B) post-UV. The results showed that knockdown of Nlp lowered the cell survival, while over-expression of Nlp significantly enhanced it, suggesting that Nlp could render cells more resistant to UVCinduced cell death. We next examined the colony formation after UVC-irradiation with doses of 0 J/m2, 2 J/m2, 4 J/m2, 6 J/m2, 8 J/m2, and found that upon UVC-irradiation, the cells with depleted expression of Nlp exhibited weaker ability of colony formation, consistent with the
Fig. 1. Nlp positively regulates UV-induced DNA damage repair. (A) Upon UV treatment, Nlp subcellular localization was changed obviously. H1299 cells were exposed to UVC 20 J/m2. After 4 hours, immunofluorescence assay was conducted. Quantification image (right) was obtained through analyzing anti-Nlp staining intensity of 500 cells. (B) Depleted Nlp levels after siRNA knockdown in H1299 were measured by real-time PCR and western blotting. (C and D) Knockdown of Nlp in H1299 reduced HCR activity (C) and removal efficiency of CPDs induced by UVC 40 J/m2 (D). (E) Expression levels of GFP-Nlp in HeLa stable cell line were detected by western blotting. (F and G) Overexpression of Nlp in HeLa cells improved HCR activity (F) and removal efficiency of CPDs induced by UVC 40 J/m2 (G). (H) In MEFs (derived from Nlp transgenic or wild type mice) examination, after UV 20 J/m2 treatment, the removal efficiency of CPDs was higher in Nlp MEFs than WT MEFs. Removal efficiency of CPDs was measured by ELISA assay. All experiments were performed at least three times and data were statistically analyzed by a two-sided t-test. *P < 0.05, **P < 0.01, ***P < 0.001 vs control. Nlp MEF, Nlp transgenic MEFs; WT MEF, wild type MEFs. Error bars indicate s.e.m.
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Fig. 2. Nlp protects cells from UV-induced cell death. (A and B) 24 hours after UVC exposure with different doses, cell survival was tested in H1299 knockdown of Nlp (A) and HeLa stable cell lines (B) respectively. The results were normalized to 0 J/m2 of each type of cells. (C and D) Colony formation ability was analyzed on 7 days after UVC treatment with indicated various doses in H1299 knockdown of Nlp (C) and HeLa stably expressing GFP-Nlp or GFP (D). Representative images (C, top and D, top) and quantification (C, bottom and D, bottom) were shown. The results were relative to 0 J/m2 of each type of cells. (E) Cell survival of MEFs derived from Nlp transgenic or wt mice was measured at 24 hours post UVC radiation with different doses. The results were relative to 0 J/m2 of each type of cells. (F) Apoptosis of Nlp MEFs (derived from Nlp transgenic mice) and wt MEFs (derived from wild type mice) induced by UVC with various doses was tested by flow cytometry with double staining 7-AAD and Annexin V at 24 h post-UVC. Representative images (F, left) and quantification (F, right) were shown. The result curves were obtained relative to 0 J/m2 of each type of cells. (G) In vivo apoptosis examination was performed by TUNEL assay in newborn Nlp transgenic and wild type mice dermal tissues after UVB 1000 J/m2 for 24 hours. The left and right images represented Nlp transgenic and wild type mice dermal tissues respectively. The white arrows indicated apoptotic cells. All experiments were conducted at least three times and data were statistically analyzed by a two-sided t-test. *P < 0.05, **P < 0.01 vs control. Error bars indicate s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observation that the cells over-expressing Nlp revealed stronger colony formation capability than the control (Fig. 2C and D). In addition to cell lines, MEFs derived from Nlp transgenic and wild type mice were employed to examine cell survival at 24 h (Fig. 2E) or 48 h (Supplementary Fig. S1C) after UVC radiation (0 J/ m2, 5 J/m2, 10 J/m2, 20 J/m2, and 40 J/m2). The results showed that Nlp transgenic MEFs had much more survival of cells than wt MEFs post-UV. The MEFs were then used to test apoptosis at 24 h after UVC with doses (0 J/m2, 5 J/m2, 10 J/m2, 20 J/m2, and 30 J/m2). Clearly, Nlp transgenic MEFs had stronger ability to resist apoptosis induced by UVC compared with the counterparts (Fig. 2F). And, this result was confirmed by western blotting assay (Supplementary Fig. S4E). Finally, we validated the findings of cell models in Nlp transgenic mice. Both Nlp transgenic and wild type newborn mice were irradiated with 1000 J/m2 UVB. At the established peak response time of 24 h after irradiation [29,30], their skin was harvested and analyzed by TUNEL assay [29]. Wt mice had appreciable numbers of TUNEL-positive keratinocytes after irradiation, while Nlp trans-
genic mice skin was more resistant to apoptosis and only rarely showed TUNEL-positive cells (Fig. 2G, white arrows). Therefore, we concluded that Nlp may reduce cell sensitivity to UV and protect cells from UV-irradiation–induced apoptosis. Nlp is translocated to the nucleus in response to UVC-irradiation As reported, nuclear import is a prerequisite for the functions of DNA repair proteins [31–34]. Interestingly, the subcellular localization of Nlp changed markedly upon UVC irradiation (Fig. 1A). Endogenous Nlp was observed in the nucleus and cytoplasm before UVC treatment. 4 hours after UVC irradiation (20 J/m2), Nlp formed marked foci within the nucleus. To detail the features of Nlp translocation, H1299 cells were irradiated with different doses of UVC and fixed at designed time points after UVC. Through immunofluorescence method, we found that endogenous Nlp positioning pattern within nucleus changed in a time and dose-dependent manner (Supplementary Fig. S1D).
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Fig. 3. Part of Nlp is translocated into the nucleus induced by UV, independently of p53. (A) 4 hours after UVC 20 J/m2 or no UV, Nlp localization was detected by immunofluorescence staining with anti-Nlp and DAPI in A549 cells. (B) Nlp could be translocated to UV-induced DNA damage sites. H1299 cells were treated by local radiation with UVC 50 J/m2 through 5 μm holes. After 30 min, double-staining immunofluorescence was conducted with anti-CPDs and anti-Nlp. (C) The mRNA and protein expression levels of Nlp were monitored at different time points after UVC 20 J/m2 by real-time PCR (left) and western blotting (right) respectively. (D) Post UV-irradiation, nuclear and cytoplasm lysates of H1299 cells were extracted using Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents kit, followed by western blotting assay with anti-Nlp, anti-LaminB (nuclear loading control protein) and β-actin. (E) Post-UVC 4 h, the nuclear, cytoplasm and whole-cell lysates of HeLa cells stably expressing GFP-Nlp (left) or GFP (right) were tested by western blotting. (F) C-terminal (1030–1382) of Nlp is responsible for its nuclear import upon UV-irradiation. HeLa cells transiently expressing myc-tag Nlp full-length or different myc-tag Nlp truncated mutants, including myc-Nlp (1–370), myc-Nlp (361–831), myc-Nlp (691–1050) and myc-Nlp (1030–1382) were treated with UVC 20 J/m2 or no UV. After 4 hours, their nuclear and whole-cell lysates were prepared for western blotting assay with anti-Myc and antiLaminB. Gray scanning was conducted with ImageJ software.
Considering that p53 plays a very important role in UV-induced DNA damage response [35,36], besides H1299 cells which did not express p53, we employed A549 cell line expressing wild type p53 to test the influence of p53 on Nlp subcellular localization. As shown in Fig. 3A, similar change of Nlp positioning happened in A549 cells after UVC, suggesting that the translocation of Nlp induced by UVirradiation may be independent of cellular p53 and cell lines. Importantly, when cells sustained local radiation with UVC 50 J/ m2 through 5 μm holes, endogenous Nlp exhibited co-localization with CPDs 30 min post-UVC (Fig. 3B). Consequently, Nlp could be translocated to UV-induced DNA damage sites, and its positioning pattern depended on cell radiation area (part or whole) and the incubation time (30 min or 4 hours) post-UV.
To investigate the expression of Nlp upon UV radiation, the total RNA and protein were analyzed at fixed time points after irradiation. We found that the expression of endogenous Nlp barely changed (Fig. 3C). Then, we prepared the nuclear and cytoplasmic lysates at predetermined time points after UVC 20 J/m2. Western blotting analysis revealed that a portion of endogenous Nlp was accumulated in the nucleus from the cytoplasm upon UV radiation (Fig. 3D). Similar results were obtained through immunofluorescence assay (Fig. 1A, right). Simultaneously, HeLa cells stably expressing GFPNlp or GFP were disposed with the same approach. Consistently, GFP-Nlp in nuclear fraction was increased, which total expression level remained constant, indicating that some exogenous Nlp was redistributed from the cytoplasm to the nucleus upon UV-irradiation
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(Fig. 3E). In conclusion, under the circumstances of UV treatment, the expression of Nlp remains unchanged; meanwhile, a part of this protein is redistributed to the nucleus. To define the region required for Nlp nuclear import, HeLa cells were transfected with full-length myc-tag Nlp and different myctag Nlp truncated mutants including myc-Nlp (1–370), myc-Nlp (361–831), myc-Nlp (691–1050) and myc-Nlp (1030–1382), followed by UVC exposure. 4 hours after treatment, nuclear lysates were analyzed. As shown in Fig. 3F, although the sections including myctag Nlp full-length, myc-Nlp (361–831), myc-Nlp (691–1050) and myc-Nlp (1030–1382) were detected in the nucleus before and after UV treatment, only full length myc-tag Nlp and myc-Nlp (1030– 1382) fragment were observed to be increased in the nucleus after UV. To confirm this result, HeLa cells were processed with the same approach followed by immunofluorescence assay, which revealed a similar result (Supplementary Fig. S2). Thus, the C-terminal (1030– 1382) of Nlp was suggested to be associated with its nuclear import induced by UVC irradiation.
Nlp interacts with XPA and ERCC1, and enhances their interaction As clarified, Nlp is translocated to the nucleus and improves UV-induced DNA repair. So we focused on the NER pathway to investigate the mechanism underlying the events. Considering that XPA is the central component of the core NER factors [7,10–16], we tested the relationship of Nlp and XPA. Through coimmunoprecipitation assay, we found that Nlp was physically associated with XPA and the association was more obvious upon UV treatment than no UV groups (Fig. 4A and B). Additionally, similar results were obtained with GST pull-down assay (Fig. 4C). Furthermore, H1299 cells exposed to UVC were processed by immunofluorescence technique and Nlp was found to be colocalized with XPA, induced by UVC radiation (Fig. 4D). All these results indicate that there is a physical interaction between Nlp and XPA. To identify which fragment of Nlp mediates its interaction with XPA, myc-tag Nlp full length or a series of myc-tag Nlp truncated
Fig. 4. Upon UV-irradiation, Nlp interacts with XPA and ERCC1, and stabilizes the complex of XPA and ERCC1. (A, B, F and G) Nlp interacts with XPA and ERCC1 in vivo. 4 hours after UVC 20 J/m2 radiation, cell lysates of H1299 were incubated with anti-Nlp, anti-XPA or anti-ERCC1 for co-immunoprecipitation assay followed by western blotting. Anti-Nlp pulled down XPA (A) and ERCC1 (F). Meanwhile, Nlp was pulled down by anti-XPA (B) and anti-ERCC1 (G) reciprocally. (C) The interaction of these three proteins was confirmed by GST pull-down in vitro. (D) After UVC 20 J/m2 or no UV for 4 hours, H1299 cells were dealt with immunofluorescence. Double-staining of antiNlp and anti-XPA revealed the co-localization of Nlp and XPA induced by UV. (E) C-terminal (1030–1382) of Nlp mediates the interaction between XPA and Nlp. HeLa cells transiently expressing myc-tag Nlp full-length or different myc-tag Nlp truncated mutants, including myc-Nlp (1–370), myc-Nlp (361–831), myc-Nlp (691–1050) and mycNlp (1030–1382) were treated with UVC 20 J/m2. After 4 hours, these cell lysates were prepared to be incubated with anti-XPA, followed by co-IP assay and western blotting. Anti-XPA pulled down myc-tag Nlp full-length and myc-Nlp (1030–1382) fragment. (H and I) Knockdown of Nlp weakens the interaction between XPA and ERCC1. AntiXPA (H) or anti-ERCC1 (I) was incubated with H1299 cell lysates, transfected with siRNAs of Nlp, after UVC 20 J/m2. Co-IP and western blotting were conducted. Upon UVC and Nlp RNAi, the complex of XPA and ERCC1 was decreased. (J and K) Over-expression of Nlp enhances the interaction between XPA and ERCC1. Anti-XPA (J) or anti-ERCC1 (K) was incubated with HeLa cell lysates stably expressing GFP-Nlp, after UVC 20 J/m2. Co-IP and western blotting were done. (L and M) Nlp does not affect XPA and ERCC1 expression. The expression levels of Nlp, XPA and ERCC1 were detected by western blotting assay in H1299 transfected with siRNAs of Nlp (L) and HeLa stably expressing GFP-Nlp (M).
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Fig. 5. Low expression level of Nlp in human skin carcinoma. Immunohistochemical assay staining with anti-Nlp was conducted in normal dermal tissue (A), cancer adjacent skin tissue (B), squamous cell carcinoma (C), basal cell carcinoma (D) and malignant melanoma (E). Original magnification, ×100 (top) and ×200 (bottom).
mutants was transfected into HeLa cells. Then 4 hours after UVC 20 J/m2 treatment, the cell lysates were prepared for co-IP with antiXPA antibody, followed by blotting with anti-Myc. The results displayed that XPA was only co-purified with the full length and the C-terminal fragment (1030–1382) of Nlp, indicating that the C-terminal (1030–1382) of Nlp was critical for its interaction with XPA (Fig. 4E). Because Nlp is a large protein with 4 tandem arrays of predicted coiled coil domains on the C terminal, which have been regarded as the mediators for protein–protein interaction, we supposed that Nlp might act as a scaffold to regulate protein-complex function. ERCC1 is another important member of the core NER factors, recruited to NER complexes by interaction with XPA [10,17]. To test the relationship of Nlp and ERCC1, H1299 cells were treated with UVC 20 J/m2. 4 h post-UVC, the whole cellular extractions were prepared to carry out the co-IP assay with anti-Nlp and antiERCC1 respectively. As shown in Fig. 4F and G, Nlp and ERCC1 could be co-purified with each other, and the interaction between Nlp and ERCC1 was greatly enhanced upon UV-irradiation. Consistently, a similar result was obtained through GST pull-down assay (Fig. 4C). Subsequently, because the interaction between ERCC1 and XPA is essential for successful NER function, the association between Nlp and the complex of XPA and ERCC1 was tested. Here, on the one hand, H1299 cells were transfected with Nlp siRNAs or control siRNAs, and then exposed to UVC 20 J/m2. The cell lysates were prepared for immunoprecipitation with anti-XPA or anti-ERCC1. As shown in Fig. 4H and I, XPA immunoprecipitated less ERCC1 in cells silenced for Nlp in contrast to the control cells and vice versa, in-
dicating that depletion of Nlp expression impaired the interaction between XPA and ERCC1. On the other hand, HeLa cells with stable expression of GFP-Nlp or GFP were assayed with similar approach and the results were shown in Fig. 4J and K. XPA could bind to more ERCC1 in GFP-Nlp cells compared with the counterparts, suggesting that high expression of Nlp was able to strengthen the interaction between XPA and ERCC1. In addition, to exclude the potential impact of Nlp on the expression levels of XPA and ERCC1, transfection and western blotting were conducted (Fig. 4L and M). The results revealed that both XPA and ERCC1 expressions were not influenced by Nlp. Low expression of Nlp is associated with human skin cancers To investigate whether Nlp abnormality was linked to human skin cancers, we analyzed Nlp protein expression in tumor tissues. Totally, an immunohistochemical approach was employed to examine 51 squamous cell carcinoma specimens, 62 basal cell carcinoma specimens, 23 malignant melanoma specimens, 17 cancer adjacent normal skin tissue samples and 7 normal dermal tissue samples. As shown in Fig. 5, low expression of Nlp was found in skin carcinomas (Fig. 5C, squamous cell carcinoma; D, basal cell carcinoma; E, malignant melanoma) compared with normal counterparts (Fig. 5A and B). The results of immunohistochemisty were summarized in Table 1. Because there was no significant difference in the Nlp expression between cancer adjacent normal skin tissue and normal dermal tissue (the results were not shown), the two groups were combined into a group. Among 51 squamous cell carcinoma samples,
Table 1 Low expression of Nlp in human skin carcinoma. Characteristic
Nlp expression (−) or (+)
Normal dermal tissue and cancer adjacent normal skin tissue Skin carcinoma Squamous cell carcinoma Basal cell carcinoma Malignant melanoma a b c
(++)
(+++)
Total case
5
10
9
24
36 53 21
14 7 2
1 2 0
51 62 23
Squamous cell carcinoma vs normal dermal tissue and cancer adjacent normal skin tissue. Basal cell carcinoma vs normal dermal tissue and cancer adjacent normal skin tissue. Malignant melanoma vs normal dermal tissue and cancer adjacent normal skin tissue.
P value
<0.001a <0.001b <0.001c
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moderate (++) and strong (+++) staining of Nlp protein was detected in 15 specimens (29.4%). Negative (−) and weak (+) staining was detected in 36 specimens (70.6%). Of the 62 basal cell carcinoma samples, moderate (++) and strong (+++) staining of Nlp protein was detected in 9 specimens (14.5%), while negative (−) and weak (+) staining was detected in 53 specimens (85.5%). And among 23 malignant melanoma specimens, moderate (++) and strong (+++) staining of Nlp protein was only detected in 2 samples (8.7%), while negative (−) and weak (+) staining was detected in 21 samples (91.3%). In contrast, of the 24 normal and cancer adjacent normal skin tissues, moderate (++) and strong (+++) staining of Nlp protein was detected in 19 samples (79.2%). Negative (−) and weak (+) staining was detected in 5 specimens (20. 8%). Through statistical analysis, the expression level of Nlp of each type of skin cancers was significantly lower than that of the normal counterparts (P < 0.001). Next, we analyzed clinical relevant variables such as age, T, N, M, clinical stage and gender. Unfortunately, little relationship was uncovered between these factors and Nlp expression in these human skin cancer samples (the results were not shown.). Discussion The genome is subjected to continuous damage and repair. Both endogenous and exogenous carcinogens can result in DNA damage and trigger multiple signal transduction pathways to slow down cell cycle progression and repair the damaged DNA to maintain the integrity of the whole genome. Among these DNA repair pathways, nucleotide excision repair (NER) is particularly attractive for its extraordinarily wide range of substrates, including UV-induced photoproducts (cyclopyrimidine dimers [CPDs], 6-4 photoproducts [(6-4)PPs]), adducts formed by environmental mutagens such as benzo[a]pyrene or various aromatic amines, certain oxidative endogenous lesions and adducts formed by cancer chemotherapeutic drugs such as cisplatin [8]. If some disruption occur in any part of DNA damage responses, the damaged DNA cannot be effectively repaired, leading to genomic instability and tumorigenesis. In the previous study by our group and others, Nlp has been identified as a BRCA1-associated centrosomal protein and plays roles in centrosome maturation, microtubule organization, mitotic spindle formation and cytokinesis during cell cycle progression [18,20,22–24,26]. However, little is known about Nlp function after genotoxic stress. In this work, we have explored a novel role of Nlp in DNA damage response to UV-irradiation. Nlp enhances DNA repair following UV-induced DNA damage and protects cells from UVinduced cell death. The mechanism by which Nlp is involved in DNA repair following UV-irradiation is investigated in this study. In addition to the observations that Nlp localizes on centrosomes and midbody to regulate centrosome maturation and cytokinesis [22,24], we have found that the expression of Nlp is not effected by UV radiation, and after UV-irradiation, parts of Nlp are redistributed into the nucleus to form foci at the DNA damage sites. We also find that some of Nlp still localizes on centrosomes upon UV treatment (Fig. 4D), indicating that the impact of Nlp on NER is likely to be independent of its role in orchestrating centrosome and cytokinesis. Furthermore, Nlp UVinduced nuclear import is dependent on its C-terminus (1030– 1382). Nucleotide excision repair (NER) pathway has been widely studied, and the core factors of this biological event have already been clear. XPA and ERCC1 are both components of core factors of the NER pathway. XPA functions through DNA damage recognition to dual excision, while ERCC1 is recruited by XPA to start the incision. And the interaction between ERCC1 and XPA is essential for successful NER function. Because Nlp is translocated to the DNA damage sites and promotes the removal of photoproducts induced by UV, it is reasonable to think that Nlp is involved in the NER
pathway. As expected, Nlp is found to interact with XPA and ERCC1 physically post-UV. Interestingly, the association between XPA and ERCC1 is obviously further strengthened when Nlp expression is upregulated, while the complex of XPA and ERCC1 is attenuated markedly when Nlp expression is depleted. In conclusion, upon UVirradiation, Nlp may act as a scaffold to interact with XPA and ERCC1 and enhance the complex of XPA and ERCC, which is required for NER after UV-induced DNA damage. Disruption of DNA repair leads to genomic instability, which is closely related to the tumor occurrence and malignant development. Ultraviolet irradiation has been linked to skin cancers. In our study, we examined the relationship between the expression level of Nlp and skin cancers. 51 squamous cell carcinoma specimens, 62 basal cell carcinoma specimens, 23 malignant melanoma specimens, 17 cancer adjacent normal skin tissue samples and 7 normal dermal tissue samples are examined. The proportion of moderate (++) and strong (+++) staining in each type of carcinomas is far less than that seen in normal skin tissues, which is statistically significant. These results suggest that low expression of Nlp is associated with human skin cancers, indicating that dysregulation of Nlp might contribute to the development of human skin cancers. Our previous studies have demonstrated that over-expression of Nlp is associated with human breast cancer, lung carcinoma [28], head and neck squamous cell carcinoma (HNSCC) [37] and ovarian cancer [38]. Consistently, transgenic mice overexpressing Nlp display spontaneous tumors in breast, ovary and testicle, and show rapid onset of radiation-induced lymphoma [28]. For these two different phenotypes, it can be thought that tumorigenesis by overexpression of Nlp might be due to disruption of microtubule organization, spindle formation and cytokinesis, while in human skin cancers, the carcinogenicity of Nlp low expression may be attributed to weakening NER activity. In conclusion, the current study demonstrates for the first time a new aspect of Nlp function in maintaining genome integrity after DNA damage. As summarized in Supplementary Fig. S3, Nlp is identified as a new protein involved in the NER pathway. After UVirradiation, some of Nlp is translocated to the nucleus from the cytoplasm depending on its C-terminal. Then, Nlp interacts with XPA and ERCC1, and enhances the complex of XPA and ERCC1 to positively modulate NER pathway activity. Because Nlp could improve the removal of CPDs, and ERCC1-XPF cleaves the 5′ end of the damage to initiate the dual incision, which is recruited to the NER complexes by XPA, we propose that Nlp might function before the excision stage. As is believed, NER could remove many types of DNA lesions including those induced by UV radiation and platinumbased therapy. High activity level of NER usually confers cells strong resistance to platinum. Therefore, Nlp might be a potential target for tumor treatment.
Acknowledgments This work is supported by the 973 National Key Fundamental Research Program of China (2015CB5539-4) and the National Natural Fund of China (81230047, 81231091).
Conflict of interest None.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.01.020.
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation Xiao-Juan Ma, Li Shang, Wei-Min Zhang, Ming-Rong Wang, Qi-Min Zhan * State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China
A R T I C L E
I N F O
Article history: Received 30 October 2015 Received in revised form 10 January 2016 Accepted 11 January 2016 Keywords: Genomic stability Nlp UV radiation NER Tumorigenesis
A B S T R A C T
Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030– 1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER. © 2016 Elsevier Ireland Ltd. All rights reserved.
Introduction Ultraviolet (UV) irradiation has been linked to skin cancers, including non-melanoma and melanoma skin cancers [1–3]. The major DNA lesions induced by UVB or by high-energy artificial UVC irradiation are cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts; (6-4) PPs]. Usually, CPDs are formed quite significantly much more than (6-4) PPs [4,5]. CPDs formation and repair are important in connection with carcinogenesis in human skin [6]. Nucleotide excision repair (NER) is the main biological event responsible for the removal of bulky, helix-distorting DNA lesions, induced by UV irradiation, environmental mutagens, and certain chemo-therapeutic agents [7,8]. The NER pathway consists of two sub-pathways of NER, global genome NER (GG-NER) and transcription-coupled NER (TC-NER), differing in damage recognition [8,9]. Both sub-pathways require the core NER factors, of which XPA is the central component as it interacts with TFIIH, RPA, XPCRAD23B, DDB2, ERCC1-XPF, and PCNA proteins [7,10–16]. ERCC1XPF is recruited to NER complexes by interaction with XPA to trigger the dual incision reaction [10,17].
* Corresponding author. Tel.: +86 10 67762694; fax: +86 10 67715058. E-mail address: [email protected] (Q.-M. Zhan). http://dx.doi.org/10.1016/j.canlet.2016.01.020 0304-3835/© 2016 Elsevier Ireland Ltd. All rights reserved.
Nlp (ninein-like protein) has been characterized as a centrosomal protein that is essential for proper mitotic events. Nlp is tethered to the centrosome by BRCA1 and is involved in microtubule organization in interphase cells by recruiting γ-TuRCs and displaced from maturing centrosome at G2/M transition to ensure mitotic spindle formation [18–22]. Its subcellular localization and protein stability are regulated by several crucial mitotic kinases, such as Plk1, Nek2, Cdc2 and Aurora B [18,20,23–26]. Deregulation of Nlp in cells results in aberrant spindle, chromosomal missegregation and multinucleus, abnormal cytokinesis and induces chromosomal instability and renders cell tumorigenesis [18,20,23,24,26]. Our previous studies have identified that Nlp interacts with BRCA1 through its C terminus, which is required for normal microtubule nucleation at interphase [22]. Since BRCA1 also functions in DNA damage response [27], we propose that Nlp might implicate in the process of DNA repair. In this work, we show that Nlp plays a role in NER after UV-induced DNA damage. More interestingly, downregulated expression of Nlp is observed in human skin carcinomas. Materials and methods H1299, A549 and HeLa cell lines were purchased from ATCC (American Type Culture Collection). They were cultured under desired medium. For UV treatment, exponentially growing cells or the cells after transfection were rinsed with phosphatebuffered saline (PBS) and irradiated with UV radiation at different doses. After UV irradiation, fresh medium was added and the cells were cultured until harvest. Detailed information is shown in the Supplementary materials.
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Results Nlp enhances DNA repair following UV-induced DNA damage In order to determine whether Nlp is involved in DNA damage response, we treated H1299 cells (lung cancer cell line) with IR or UV with appropiate doses. There was little change of subcellular localization of Nlp in the case of IR which mainly caused double strand breaks (DSBs) (data not shown). However, after UV radiation, the localization of Nlp changed obviously (Fig. 1A). Then we asked whether Nlp contributes to photoproducts excision. Host cell reactivation (HCR) assay and the removal of CPDs assay by ELISA were employed. Upon knockdown of Nlp expression in H1299 cell line (Fig. 1B), the HCR activity and the rate of CPDs removal were significantly attenuated compared with the control groups (Fig. 1C and D). Similarly, both the HCR activity and the removal of CPDs were more powerful in HeLa cells (cervical cancer cell line) reconstituted with pEGFP-C3-Nlp and KYSE30 cells (esophageal squamous cell carcinoma cell line) transfected with pcDNA3.1(+)-Nlp than their counterparts (Fig. 1E–G and Supplementary Fig. S4A–C). Consistently, when the removal of CPDs was measured in MEFs derived
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from Nlp transgenic [28] and wild type mice, similiar results were obtained (Fig. 1H). Collectively, the findings indicate that Nlp is involved in the DNA repair process and probably regulates nucleotide excision repair (NER) pathway following UV-radiation. Nlp protects cells from UV radiation-induced cell death Given that Nlp promotes NER activity, we next explored its physiological functions. H1299 cells transfected with Nlp siRNAs, HeLa and KYSE30 cells overexpressing Nlp were treated by UVC radiation with the indicated doses (0 J/m2, 5 J/m2, 10 J/m2, 20 J/m2, 40 J/ m2). Cell survival was then measured at 24 hours (Fig. 2A and B and Supplementary Fig. S4D) or 42 hours (Supplementary Fig. S1A and B) post-UV. The results showed that knockdown of Nlp lowered the cell survival, while over-expression of Nlp significantly enhanced it, suggesting that Nlp could render cells more resistant to UVCinduced cell death. We next examined the colony formation after UVC-irradiation with doses of 0 J/m2, 2 J/m2, 4 J/m2, 6 J/m2, 8 J/m2, and found that upon UVC-irradiation, the cells with depleted expression of Nlp exhibited weaker ability of colony formation, consistent with the
Fig. 1. Nlp positively regulates UV-induced DNA damage repair. (A) Upon UV treatment, Nlp subcellular localization was changed obviously. H1299 cells were exposed to UVC 20 J/m2. After 4 hours, immunofluorescence assay was conducted. Quantification image (right) was obtained through analyzing anti-Nlp staining intensity of 500 cells. (B) Depleted Nlp levels after siRNA knockdown in H1299 were measured by real-time PCR and western blotting. (C and D) Knockdown of Nlp in H1299 reduced HCR activity (C) and removal efficiency of CPDs induced by UVC 40 J/m2 (D). (E) Expression levels of GFP-Nlp in HeLa stable cell line were detected by western blotting. (F and G) Overexpression of Nlp in HeLa cells improved HCR activity (F) and removal efficiency of CPDs induced by UVC 40 J/m2 (G). (H) In MEFs (derived from Nlp transgenic or wild type mice) examination, after UV 20 J/m2 treatment, the removal efficiency of CPDs was higher in Nlp MEFs than WT MEFs. Removal efficiency of CPDs was measured by ELISA assay. All experiments were performed at least three times and data were statistically analyzed by a two-sided t-test. *P < 0.05, **P < 0.01, ***P < 0.001 vs control. Nlp MEF, Nlp transgenic MEFs; WT MEF, wild type MEFs. Error bars indicate s.e.m.
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Fig. 2. Nlp protects cells from UV-induced cell death. (A and B) 24 hours after UVC exposure with different doses, cell survival was tested in H1299 knockdown of Nlp (A) and HeLa stable cell lines (B) respectively. The results were normalized to 0 J/m2 of each type of cells. (C and D) Colony formation ability was analyzed on 7 days after UVC treatment with indicated various doses in H1299 knockdown of Nlp (C) and HeLa stably expressing GFP-Nlp or GFP (D). Representative images (C, top and D, top) and quantification (C, bottom and D, bottom) were shown. The results were relative to 0 J/m2 of each type of cells. (E) Cell survival of MEFs derived from Nlp transgenic or wt mice was measured at 24 hours post UVC radiation with different doses. The results were relative to 0 J/m2 of each type of cells. (F) Apoptosis of Nlp MEFs (derived from Nlp transgenic mice) and wt MEFs (derived from wild type mice) induced by UVC with various doses was tested by flow cytometry with double staining 7-AAD and Annexin V at 24 h post-UVC. Representative images (F, left) and quantification (F, right) were shown. The result curves were obtained relative to 0 J/m2 of each type of cells. (G) In vivo apoptosis examination was performed by TUNEL assay in newborn Nlp transgenic and wild type mice dermal tissues after UVB 1000 J/m2 for 24 hours. The left and right images represented Nlp transgenic and wild type mice dermal tissues respectively. The white arrows indicated apoptotic cells. All experiments were conducted at least three times and data were statistically analyzed by a two-sided t-test. *P < 0.05, **P < 0.01 vs control. Error bars indicate s.e.m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observation that the cells over-expressing Nlp revealed stronger colony formation capability than the control (Fig. 2C and D). In addition to cell lines, MEFs derived from Nlp transgenic and wild type mice were employed to examine cell survival at 24 h (Fig. 2E) or 48 h (Supplementary Fig. S1C) after UVC radiation (0 J/ m2, 5 J/m2, 10 J/m2, 20 J/m2, and 40 J/m2). The results showed that Nlp transgenic MEFs had much more survival of cells than wt MEFs post-UV. The MEFs were then used to test apoptosis at 24 h after UVC with doses (0 J/m2, 5 J/m2, 10 J/m2, 20 J/m2, and 30 J/m2). Clearly, Nlp transgenic MEFs had stronger ability to resist apoptosis induced by UVC compared with the counterparts (Fig. 2F). And, this result was confirmed by western blotting assay (Supplementary Fig. S4E). Finally, we validated the findings of cell models in Nlp transgenic mice. Both Nlp transgenic and wild type newborn mice were irradiated with 1000 J/m2 UVB. At the established peak response time of 24 h after irradiation [29,30], their skin was harvested and analyzed by TUNEL assay [29]. Wt mice had appreciable numbers of TUNEL-positive keratinocytes after irradiation, while Nlp trans-
genic mice skin was more resistant to apoptosis and only rarely showed TUNEL-positive cells (Fig. 2G, white arrows). Therefore, we concluded that Nlp may reduce cell sensitivity to UV and protect cells from UV-irradiation–induced apoptosis. Nlp is translocated to the nucleus in response to UVC-irradiation As reported, nuclear import is a prerequisite for the functions of DNA repair proteins [31–34]. Interestingly, the subcellular localization of Nlp changed markedly upon UVC irradiation (Fig. 1A). Endogenous Nlp was observed in the nucleus and cytoplasm before UVC treatment. 4 hours after UVC irradiation (20 J/m2), Nlp formed marked foci within the nucleus. To detail the features of Nlp translocation, H1299 cells were irradiated with different doses of UVC and fixed at designed time points after UVC. Through immunofluorescence method, we found that endogenous Nlp positioning pattern within nucleus changed in a time and dose-dependent manner (Supplementary Fig. S1D).
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Fig. 3. Part of Nlp is translocated into the nucleus induced by UV, independently of p53. (A) 4 hours after UVC 20 J/m2 or no UV, Nlp localization was detected by immunofluorescence staining with anti-Nlp and DAPI in A549 cells. (B) Nlp could be translocated to UV-induced DNA damage sites. H1299 cells were treated by local radiation with UVC 50 J/m2 through 5 μm holes. After 30 min, double-staining immunofluorescence was conducted with anti-CPDs and anti-Nlp. (C) The mRNA and protein expression levels of Nlp were monitored at different time points after UVC 20 J/m2 by real-time PCR (left) and western blotting (right) respectively. (D) Post UV-irradiation, nuclear and cytoplasm lysates of H1299 cells were extracted using Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents kit, followed by western blotting assay with anti-Nlp, anti-LaminB (nuclear loading control protein) and β-actin. (E) Post-UVC 4 h, the nuclear, cytoplasm and whole-cell lysates of HeLa cells stably expressing GFP-Nlp (left) or GFP (right) were tested by western blotting. (F) C-terminal (1030–1382) of Nlp is responsible for its nuclear import upon UV-irradiation. HeLa cells transiently expressing myc-tag Nlp full-length or different myc-tag Nlp truncated mutants, including myc-Nlp (1–370), myc-Nlp (361–831), myc-Nlp (691–1050) and myc-Nlp (1030–1382) were treated with UVC 20 J/m2 or no UV. After 4 hours, their nuclear and whole-cell lysates were prepared for western blotting assay with anti-Myc and antiLaminB. Gray scanning was conducted with ImageJ software.
Considering that p53 plays a very important role in UV-induced DNA damage response [35,36], besides H1299 cells which did not express p53, we employed A549 cell line expressing wild type p53 to test the influence of p53 on Nlp subcellular localization. As shown in Fig. 3A, similar change of Nlp positioning happened in A549 cells after UVC, suggesting that the translocation of Nlp induced by UVirradiation may be independent of cellular p53 and cell lines. Importantly, when cells sustained local radiation with UVC 50 J/ m2 through 5 μm holes, endogenous Nlp exhibited co-localization with CPDs 30 min post-UVC (Fig. 3B). Consequently, Nlp could be translocated to UV-induced DNA damage sites, and its positioning pattern depended on cell radiation area (part or whole) and the incubation time (30 min or 4 hours) post-UV.
To investigate the expression of Nlp upon UV radiation, the total RNA and protein were analyzed at fixed time points after irradiation. We found that the expression of endogenous Nlp barely changed (Fig. 3C). Then, we prepared the nuclear and cytoplasmic lysates at predetermined time points after UVC 20 J/m2. Western blotting analysis revealed that a portion of endogenous Nlp was accumulated in the nucleus from the cytoplasm upon UV radiation (Fig. 3D). Similar results were obtained through immunofluorescence assay (Fig. 1A, right). Simultaneously, HeLa cells stably expressing GFPNlp or GFP were disposed with the same approach. Consistently, GFP-Nlp in nuclear fraction was increased, which total expression level remained constant, indicating that some exogenous Nlp was redistributed from the cytoplasm to the nucleus upon UV-irradiation
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(Fig. 3E). In conclusion, under the circumstances of UV treatment, the expression of Nlp remains unchanged; meanwhile, a part of this protein is redistributed to the nucleus. To define the region required for Nlp nuclear import, HeLa cells were transfected with full-length myc-tag Nlp and different myctag Nlp truncated mutants including myc-Nlp (1–370), myc-Nlp (361–831), myc-Nlp (691–1050) and myc-Nlp (1030–1382), followed by UVC exposure. 4 hours after treatment, nuclear lysates were analyzed. As shown in Fig. 3F, although the sections including myctag Nlp full-length, myc-Nlp (361–831), myc-Nlp (691–1050) and myc-Nlp (1030–1382) were detected in the nucleus before and after UV treatment, only full length myc-tag Nlp and myc-Nlp (1030– 1382) fragment were observed to be increased in the nucleus after UV. To confirm this result, HeLa cells were processed with the same approach followed by immunofluorescence assay, which revealed a similar result (Supplementary Fig. S2). Thus, the C-terminal (1030– 1382) of Nlp was suggested to be associated with its nuclear import induced by UVC irradiation.
Nlp interacts with XPA and ERCC1, and enhances their interaction As clarified, Nlp is translocated to the nucleus and improves UV-induced DNA repair. So we focused on the NER pathway to investigate the mechanism underlying the events. Considering that XPA is the central component of the core NER factors [7,10–16], we tested the relationship of Nlp and XPA. Through coimmunoprecipitation assay, we found that Nlp was physically associated with XPA and the association was more obvious upon UV treatment than no UV groups (Fig. 4A and B). Additionally, similar results were obtained with GST pull-down assay (Fig. 4C). Furthermore, H1299 cells exposed to UVC were processed by immunofluorescence technique and Nlp was found to be colocalized with XPA, induced by UVC radiation (Fig. 4D). All these results indicate that there is a physical interaction between Nlp and XPA. To identify which fragment of Nlp mediates its interaction with XPA, myc-tag Nlp full length or a series of myc-tag Nlp truncated
Fig. 4. Upon UV-irradiation, Nlp interacts with XPA and ERCC1, and stabilizes the complex of XPA and ERCC1. (A, B, F and G) Nlp interacts with XPA and ERCC1 in vivo. 4 hours after UVC 20 J/m2 radiation, cell lysates of H1299 were incubated with anti-Nlp, anti-XPA or anti-ERCC1 for co-immunoprecipitation assay followed by western blotting. Anti-Nlp pulled down XPA (A) and ERCC1 (F). Meanwhile, Nlp was pulled down by anti-XPA (B) and anti-ERCC1 (G) reciprocally. (C) The interaction of these three proteins was confirmed by GST pull-down in vitro. (D) After UVC 20 J/m2 or no UV for 4 hours, H1299 cells were dealt with immunofluorescence. Double-staining of antiNlp and anti-XPA revealed the co-localization of Nlp and XPA induced by UV. (E) C-terminal (1030–1382) of Nlp mediates the interaction between XPA and Nlp. HeLa cells transiently expressing myc-tag Nlp full-length or different myc-tag Nlp truncated mutants, including myc-Nlp (1–370), myc-Nlp (361–831), myc-Nlp (691–1050) and mycNlp (1030–1382) were treated with UVC 20 J/m2. After 4 hours, these cell lysates were prepared to be incubated with anti-XPA, followed by co-IP assay and western blotting. Anti-XPA pulled down myc-tag Nlp full-length and myc-Nlp (1030–1382) fragment. (H and I) Knockdown of Nlp weakens the interaction between XPA and ERCC1. AntiXPA (H) or anti-ERCC1 (I) was incubated with H1299 cell lysates, transfected with siRNAs of Nlp, after UVC 20 J/m2. Co-IP and western blotting were conducted. Upon UVC and Nlp RNAi, the complex of XPA and ERCC1 was decreased. (J and K) Over-expression of Nlp enhances the interaction between XPA and ERCC1. Anti-XPA (J) or anti-ERCC1 (K) was incubated with HeLa cell lysates stably expressing GFP-Nlp, after UVC 20 J/m2. Co-IP and western blotting were done. (L and M) Nlp does not affect XPA and ERCC1 expression. The expression levels of Nlp, XPA and ERCC1 were detected by western blotting assay in H1299 transfected with siRNAs of Nlp (L) and HeLa stably expressing GFP-Nlp (M).
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Fig. 5. Low expression level of Nlp in human skin carcinoma. Immunohistochemical assay staining with anti-Nlp was conducted in normal dermal tissue (A), cancer adjacent skin tissue (B), squamous cell carcinoma (C), basal cell carcinoma (D) and malignant melanoma (E). Original magnification, ×100 (top) and ×200 (bottom).
mutants was transfected into HeLa cells. Then 4 hours after UVC 20 J/m2 treatment, the cell lysates were prepared for co-IP with antiXPA antibody, followed by blotting with anti-Myc. The results displayed that XPA was only co-purified with the full length and the C-terminal fragment (1030–1382) of Nlp, indicating that the C-terminal (1030–1382) of Nlp was critical for its interaction with XPA (Fig. 4E). Because Nlp is a large protein with 4 tandem arrays of predicted coiled coil domains on the C terminal, which have been regarded as the mediators for protein–protein interaction, we supposed that Nlp might act as a scaffold to regulate protein-complex function. ERCC1 is another important member of the core NER factors, recruited to NER complexes by interaction with XPA [10,17]. To test the relationship of Nlp and ERCC1, H1299 cells were treated with UVC 20 J/m2. 4 h post-UVC, the whole cellular extractions were prepared to carry out the co-IP assay with anti-Nlp and antiERCC1 respectively. As shown in Fig. 4F and G, Nlp and ERCC1 could be co-purified with each other, and the interaction between Nlp and ERCC1 was greatly enhanced upon UV-irradiation. Consistently, a similar result was obtained through GST pull-down assay (Fig. 4C). Subsequently, because the interaction between ERCC1 and XPA is essential for successful NER function, the association between Nlp and the complex of XPA and ERCC1 was tested. Here, on the one hand, H1299 cells were transfected with Nlp siRNAs or control siRNAs, and then exposed to UVC 20 J/m2. The cell lysates were prepared for immunoprecipitation with anti-XPA or anti-ERCC1. As shown in Fig. 4H and I, XPA immunoprecipitated less ERCC1 in cells silenced for Nlp in contrast to the control cells and vice versa, in-
dicating that depletion of Nlp expression impaired the interaction between XPA and ERCC1. On the other hand, HeLa cells with stable expression of GFP-Nlp or GFP were assayed with similar approach and the results were shown in Fig. 4J and K. XPA could bind to more ERCC1 in GFP-Nlp cells compared with the counterparts, suggesting that high expression of Nlp was able to strengthen the interaction between XPA and ERCC1. In addition, to exclude the potential impact of Nlp on the expression levels of XPA and ERCC1, transfection and western blotting were conducted (Fig. 4L and M). The results revealed that both XPA and ERCC1 expressions were not influenced by Nlp. Low expression of Nlp is associated with human skin cancers To investigate whether Nlp abnormality was linked to human skin cancers, we analyzed Nlp protein expression in tumor tissues. Totally, an immunohistochemical approach was employed to examine 51 squamous cell carcinoma specimens, 62 basal cell carcinoma specimens, 23 malignant melanoma specimens, 17 cancer adjacent normal skin tissue samples and 7 normal dermal tissue samples. As shown in Fig. 5, low expression of Nlp was found in skin carcinomas (Fig. 5C, squamous cell carcinoma; D, basal cell carcinoma; E, malignant melanoma) compared with normal counterparts (Fig. 5A and B). The results of immunohistochemisty were summarized in Table 1. Because there was no significant difference in the Nlp expression between cancer adjacent normal skin tissue and normal dermal tissue (the results were not shown), the two groups were combined into a group. Among 51 squamous cell carcinoma samples,
Table 1 Low expression of Nlp in human skin carcinoma. Characteristic
Nlp expression (−) or (+)
Normal dermal tissue and cancer adjacent normal skin tissue Skin carcinoma Squamous cell carcinoma Basal cell carcinoma Malignant melanoma a b c
(++)
(+++)
Total case
5
10
9
24
36 53 21
14 7 2
1 2 0
51 62 23
Squamous cell carcinoma vs normal dermal tissue and cancer adjacent normal skin tissue. Basal cell carcinoma vs normal dermal tissue and cancer adjacent normal skin tissue. Malignant melanoma vs normal dermal tissue and cancer adjacent normal skin tissue.
P value
<0.001a <0.001b <0.001c
220
X.-J. Ma et al./Cancer Letters 373 (2016) 214–221
moderate (++) and strong (+++) staining of Nlp protein was detected in 15 specimens (29.4%). Negative (−) and weak (+) staining was detected in 36 specimens (70.6%). Of the 62 basal cell carcinoma samples, moderate (++) and strong (+++) staining of Nlp protein was detected in 9 specimens (14.5%), while negative (−) and weak (+) staining was detected in 53 specimens (85.5%). And among 23 malignant melanoma specimens, moderate (++) and strong (+++) staining of Nlp protein was only detected in 2 samples (8.7%), while negative (−) and weak (+) staining was detected in 21 samples (91.3%). In contrast, of the 24 normal and cancer adjacent normal skin tissues, moderate (++) and strong (+++) staining of Nlp protein was detected in 19 samples (79.2%). Negative (−) and weak (+) staining was detected in 5 specimens (20. 8%). Through statistical analysis, the expression level of Nlp of each type of skin cancers was significantly lower than that of the normal counterparts (P < 0.001). Next, we analyzed clinical relevant variables such as age, T, N, M, clinical stage and gender. Unfortunately, little relationship was uncovered between these factors and Nlp expression in these human skin cancer samples (the results were not shown.). Discussion The genome is subjected to continuous damage and repair. Both endogenous and exogenous carcinogens can result in DNA damage and trigger multiple signal transduction pathways to slow down cell cycle progression and repair the damaged DNA to maintain the integrity of the whole genome. Among these DNA repair pathways, nucleotide excision repair (NER) is particularly attractive for its extraordinarily wide range of substrates, including UV-induced photoproducts (cyclopyrimidine dimers [CPDs], 6-4 photoproducts [(6-4)PPs]), adducts formed by environmental mutagens such as benzo[a]pyrene or various aromatic amines, certain oxidative endogenous lesions and adducts formed by cancer chemotherapeutic drugs such as cisplatin [8]. If some disruption occur in any part of DNA damage responses, the damaged DNA cannot be effectively repaired, leading to genomic instability and tumorigenesis. In the previous study by our group and others, Nlp has been identified as a BRCA1-associated centrosomal protein and plays roles in centrosome maturation, microtubule organization, mitotic spindle formation and cytokinesis during cell cycle progression [18,20,22–24,26]. However, little is known about Nlp function after genotoxic stress. In this work, we have explored a novel role of Nlp in DNA damage response to UV-irradiation. Nlp enhances DNA repair following UV-induced DNA damage and protects cells from UVinduced cell death. The mechanism by which Nlp is involved in DNA repair following UV-irradiation is investigated in this study. In addition to the observations that Nlp localizes on centrosomes and midbody to regulate centrosome maturation and cytokinesis [22,24], we have found that the expression of Nlp is not effected by UV radiation, and after UV-irradiation, parts of Nlp are redistributed into the nucleus to form foci at the DNA damage sites. We also find that some of Nlp still localizes on centrosomes upon UV treatment (Fig. 4D), indicating that the impact of Nlp on NER is likely to be independent of its role in orchestrating centrosome and cytokinesis. Furthermore, Nlp UVinduced nuclear import is dependent on its C-terminus (1030– 1382). Nucleotide excision repair (NER) pathway has been widely studied, and the core factors of this biological event have already been clear. XPA and ERCC1 are both components of core factors of the NER pathway. XPA functions through DNA damage recognition to dual excision, while ERCC1 is recruited by XPA to start the incision. And the interaction between ERCC1 and XPA is essential for successful NER function. Because Nlp is translocated to the DNA damage sites and promotes the removal of photoproducts induced by UV, it is reasonable to think that Nlp is involved in the NER
pathway. As expected, Nlp is found to interact with XPA and ERCC1 physically post-UV. Interestingly, the association between XPA and ERCC1 is obviously further strengthened when Nlp expression is upregulated, while the complex of XPA and ERCC1 is attenuated markedly when Nlp expression is depleted. In conclusion, upon UVirradiation, Nlp may act as a scaffold to interact with XPA and ERCC1 and enhance the complex of XPA and ERCC, which is required for NER after UV-induced DNA damage. Disruption of DNA repair leads to genomic instability, which is closely related to the tumor occurrence and malignant development. Ultraviolet irradiation has been linked to skin cancers. In our study, we examined the relationship between the expression level of Nlp and skin cancers. 51 squamous cell carcinoma specimens, 62 basal cell carcinoma specimens, 23 malignant melanoma specimens, 17 cancer adjacent normal skin tissue samples and 7 normal dermal tissue samples are examined. The proportion of moderate (++) and strong (+++) staining in each type of carcinomas is far less than that seen in normal skin tissues, which is statistically significant. These results suggest that low expression of Nlp is associated with human skin cancers, indicating that dysregulation of Nlp might contribute to the development of human skin cancers. Our previous studies have demonstrated that over-expression of Nlp is associated with human breast cancer, lung carcinoma [28], head and neck squamous cell carcinoma (HNSCC) [37] and ovarian cancer [38]. Consistently, transgenic mice overexpressing Nlp display spontaneous tumors in breast, ovary and testicle, and show rapid onset of radiation-induced lymphoma [28]. For these two different phenotypes, it can be thought that tumorigenesis by overexpression of Nlp might be due to disruption of microtubule organization, spindle formation and cytokinesis, while in human skin cancers, the carcinogenicity of Nlp low expression may be attributed to weakening NER activity. In conclusion, the current study demonstrates for the first time a new aspect of Nlp function in maintaining genome integrity after DNA damage. As summarized in Supplementary Fig. S3, Nlp is identified as a new protein involved in the NER pathway. After UVirradiation, some of Nlp is translocated to the nucleus from the cytoplasm depending on its C-terminal. Then, Nlp interacts with XPA and ERCC1, and enhances the complex of XPA and ERCC1 to positively modulate NER pathway activity. Because Nlp could improve the removal of CPDs, and ERCC1-XPF cleaves the 5′ end of the damage to initiate the dual incision, which is recruited to the NER complexes by XPA, we propose that Nlp might function before the excision stage. As is believed, NER could remove many types of DNA lesions including those induced by UV radiation and platinumbased therapy. High activity level of NER usually confers cells strong resistance to platinum. Therefore, Nlp might be a potential target for tumor treatment.
Acknowledgments This work is supported by the 973 National Key Fundamental Research Program of China (2015CB5539-4) and the National Natural Fund of China (81230047, 81231091).
Conflict of interest None.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.01.020.
X.-J. Ma et al./Cancer Letters 373 (2016) 214–221
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