Qi-En Wang

Thymoquinone induces apoptosis through activation of caspase-8 and mitochondrial events in p53-null myeloblastic leukemia HL-60 cells

Thymoquinone (TQ), the major biologically active component isolated from a traditional medicinal herb, Nigella sativa Linn, is a potential chemopreventive and chemotherapeutic compound. Despite the promising antineoplastic activities of TQ, the molecular mechanism of its pharmacologic effects is poorly understood. Here, we report that TQ exhibits antiproliferative effect, induces apoptosis, disrupts mitochondrial membrane potential and triggers the activation of caspases 8, 9 and 3 in myeloblastic leukemia HL‐60 cells. The apoptosis induced by TQ was inhibited by a general caspase inhibitor, z‐VAD‐FMK; a caspase‐3‐specific inhibitor, z‐DEVD‐FMK; as well as a caspase‐8‐specific inhibitor, z‐IETD‐FMK. Moreover, the caspase‐8 inhibitor blocked the TQ‐induced activation of caspase‐3, PARP cleavage and the release of cytochrome c from mitochondria into the cytoplasm. In addition, TQ treatment of HL‐60 cells caused a marked increase in Bax/Bcl2 ratios due to upregulation of Bax and downregulation of Bcl2 proteins. These results indicate that TQ‐induced apoptosis is associated with the activation of caspases 8, 9 and 3, with caspase‐8 acting as an upstream activator. Activated caspase‐8 initiates the release of cytochrome c during TQ‐induced apoptosis. Overall, these results offer a potential mechanism for TQ‐induced apoptosis in p53‐null HL‐60 cancer cells.

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein.

Cullin 4A-mediated Proteolysis of DDB2 Protein at DNA Damage Sites Regulates in Vivo Lesion Recognition by XPC

Xeroderma pigmentosum (XP) complementation group E gene product, damaged DNA-binding protein 2 (DDB2), is a subunit of the DDB heterodimeric protein complex with high specificity for binding to a variety of DNA helix-distorting lesions. DDB is believed to play a role in the initial step of damage recognition in mammalian nucleotide excision repair (NER) of ultraviolet light (UV)-induced photolesions. It has been shown that DDB2 is rapidly degraded after cellular UV irradiation. However, the relevance of DDB2 degradation to its functionality in NER is still unknown. Here, we have provided evidence that Cullin 4A (CUL-4A), a key component of CUL-4A-based ubiquitin ligase, mediates DDB2 degradation at the damage sites and regulates the recruitment of XPC and the repair of cyclobutane pyrimidine dimers. We have shown that CUL-4A can be identified in a UV-responsive protein complex containing both DDB subunits. CUL-4A was visualized in localized UV-irradiated sites together with DDB2 and XPC. Degradation of DDB2 could be blocked by silencing CUL-4A using small interference RNA or by treating cells with proteasome inhibitor MG132. This blockage resulted in prolonged retention of DDB2 at the subnuclear DNA damage foci within micropore irradiated cells. Knock down of CUL-4A also decreased recruitment of the damage recognition factor, XPC, to the damaged foci and concomitantly reduced the removal of cyclobutane pyrimidine dimers from the entire genome. These results suggest that CUL-4A mediates the proteolytic degradation of DDB2 and that this degradation event, initiated at the lesion sites, regulates damage recognition by XPC during the early steps of NER.

Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.

DNA damage binding protein component DDB1 participates in nucleotide excision repair through DDB2 DNA-binding and cullin 4A ubiquitin ligase activity.

Functional defect in DNA damage binding (DDB) activity has a direct relationship to decreased nucleotide excision repair (NER) and increased susceptibility to cancer. DDB forms a complex with cullin 4A (Cul4A), which is now known to ubiquitylate DDB2, XPC, and histone H2A. However, the exact role of DDB1 in NER is unclear. In this study, we show that DDB1 knockdown in human cells impaired their ability to efficiently repair UV-induced cyclobutane pyrimidine dimers (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear protein fractionation and chromatin association analysis revealed that upon irradiation, DDB1 protein is translocated from a loosely bound to a tightly bound in vivo chromatin fraction and the DDB1 translocation required the participation of functional DDB2 protein. DDB1 knockdown also affected the translocation of Cul4A component to the tightly bound form in UV-damaged chromatin in vivo as well as its recruitment to the locally damaged nuclear foci in situ. However, DDB1 knockdown had no effect on DNA damage binding capacity of DDB2. The data indicated that DDB2 can bind to damaged DNA in vivo as a monomer, whereas Cul4A recruitment to damage sites depends on the fully assembled complex. Our data also showed that DDB1 is required for the UV-induced DDB2 ubiquitylation and degradation. In summary, the results suggest that (a) DDB1 is critical for efficient NER of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase activity to the damaged sites for successful commencement of lesion processing by NER.

Modulation of Nucleotide Excision Repair by Mammalian SWI/SNF Chromatin-remodeling Complex

Accessibility within chromatin is an important factor in the prompt removal of UV-induced DNA damage by nucleotide excision repair (NER). Chromatin remodeling by the SWI/SNF complex has been shown to play an important modulating role in NER in vitro and yeast in vivo. Nevertheless, the molecular basis of cross-talk between SWI/SNF and NER in mammalian cells is not fully understood. Here, we show that knockdown of Brg1, the ATPase subunit of SWI/SNF, negatively affects the elimination of cyclobutane pyrimidine dimers (CPD), but not of pyrimidine (6, 4)pyrimidone photoproducts (6-4PP) following UV irradiation of mammalian cells. Brg1-deficient cells exhibit a lower chromatin relaxation as well as impaired recruitment of downstream NER factors, XPG and PCNA, to UV lesions. However, the assembly of upstream NER factors, DDB2 and XPC, at the damage site was unaffected by Brg1 knockdown. Interestingly, Brg1 interacts with XPC within chromatin and is recruited to UV-damaged sites in a DDB2- and XPC-dependent manner. Also, postirradiation decrease of XPC levels occurred more rapidly in Brg1-deficient than normal cells. Conversely, XPC transcription remained unaltered upon Brg1 knockdown indicating that Brg1 affects the stability of XPC protein following irradiation. Thus, Brg1 facilitates different stages of NER by initially modulating UV-induced chromatin relaxation and stabilizing XPC at the damage sites, and subsequently stimulating the recruitment of XPG and PCNA to successfully culminate the repair.

The ubiquitin–proteasome system regulates p53-mediated transcription at p21waf1 promoter

The ubiquitin (Ub)–proteasome system (UPS) promotes the proteasomal degradation of target proteins by decorating them with Ub labels. Emerging evidence indicates a role of UPS in regulating gene transcription. In this study, we provided evidence for the involvement of UPS in the transcriptional activation function of tumor suppressor p53. We showed that both ubiquitylation and proteasomal functions are required for efficient transcription mediated by p53. Disruption of transcription by actinomycin D, 5,6-dichloro-1-β-D-ribofuranosyl-benzimadazole or α-amanitin leads to accumulation of cellular p53 protein. Proteasome inhibition by MG132 increases the occupancy of p53 protein at p53-responsive p21waf1 promoter. In addition, the Sug-1 component of 19S proteasome physically interacts with p53 in vitro and in vivo. Moreover, in response to ultraviolet-induced DNA damage, both the 19S proteasomal components, Sug1 and S1, are recruited to p21waf1 promoter region in a kinetic pattern similar to that of p53. These results suggested that UPS positively regulates p53-mediated transcription at p21waf1 promoter.

CAR-Engineered NK Cells Targeting Wild-Type EGFR and EGFRvIII Enhance Killing of Glioblastoma and Patient-Derived Glioblastoma Stem Cells

Glioblastoma (GB) remains the most aggressive primary brain malignancy. Adoptive transfer of chimeric antigen receptor (CAR)-modified immune cells has emerged as a promising anti-cancer approach, yet the potential utility of CAR-engineered natural killer (NK) cells to treat GB has not been explored. Tumors from approximately 50% of GB patients express wild-type EGFR (wtEGFR) and in fewer cases express both wtEGFR and the mutant form EGFRvIII; however, previously reported CAR T cell studies only focus on targeting EGFRvIII. Here we explore whether both wtEGFR and EGFRvIII can be effectively targeted by CAR-redirected NK cells to treat GB. We transduced human NK cell lines NK-92 and NKL, and primary NK cells with a lentiviral construct harboring a second generation CAR targeting both wtEGFR and EGFRvIII and evaluated the anti-GB efficacy of EGFR-CAR-modified NK cells. EGFR-CAR-engineered NK cells displayed enhanced cytolytic capability and IFN-γ production when co-cultured with GB cells or patient-derived GB stem cells in an EGFR-dependent manner. In two orthotopic GB xenograft mouse models, intracranial administration of NK-92-EGFR-CAR cells resulted in efficient suppression of tumor growth and significantly prolonged the tumor-bearing mice survival. These findings support intracranial administration of NK-92-EGFR-CAR cells represents a promising clinical strategy to treat GB.

Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia

Bioactive components from dietary supplements such as curcumin may represent attractive agents for cancer prevention or treatment. DNA methylation plays a critical role in acute myeloid leukemia (AML) development, and presents an excellent target for treatment of this disease. However, it remains largely unknown how curcumin, a component of the popular Indian spice turmeric, plays a role in DNA hypomethylation to reactivate silenced tumor suppressor genes and to present a potential treatment option for AML. Here we show that curcumin down-regulates DNMT1 expression in AML cell lines, both in vitro and in vivo, and in primary AML cells ex vivo. Mechanistically, curcumin reduced the expression of positive regulators of DNMT1, p65 and Sp1, which correlated with a reduction in binding of these transcription factors to the DNMT1 promoter in AML cell lines. This curcumin-mediated down-regulation of DNMT1 expression was concomitant with p15INK4B tumor suppressor gene reactivation, hypomethylation of the p15INK4B promoter, G1 cell cycle arrest, and induction of tumor cell apoptosis in vitro. In mice implanted with the human AML MV4–11 cell line, administration of curcumin resulted in remarkable suppression of AML tumor growth. Collectively, our data indicate that curcumin shows promise as a potential treatment for AML, and our findings provide a basis for future studies to test the clinical efficacy of curcumin – whether used as a single agent or as an adjuvant – for AML treatment.

Genetic Modification of T Cells Redirected toward CS1 Enhances Eradication of Myeloma Cells

Purpose: Our goal is to test whether CS1 could be targeted by chimeric antigen receptor (CAR) T cells to treat multiple myeloma (MM). Experimental Design: We generated a retroviral construct of a CS1-specific CAR and engineered primary human T cells expressing the CAR. We then tested the capacity of CS1–CAR T cells to eradicate human MM tumor cells in vitro, ex vivo, and in vivo using orthotopic MM xenograft mouse models. Results: In vitro, compared with mock-transduced T cells, upon recognizing CS1-positive MM cells, CS1–CAR-transduced T cells secreted more IFN-γ as well as interleukin (IL)-2, expressed higher levels of the activation marker CD69, showed higher capacity for degranulation, and displayed enhanced cytotoxicity. Ectopically forced expression of CS1 in MM cells with low CS1 expression enhanced recognition and killing by CAR T cells. Ex vivo, CS1–CAR T cells also showed similarly enhanced activities when responding to primary MM cells. More importantly, in orthotopic MM xenograft mouse models, adoptive transfer of human primary T cells expressing CS1–CAR efficiently suppressed the growth of human MM.1S and IM9 myeloma cells and significantly prolonged mouse survival. Conclusions: CS1 is a promising antigen that can be targeted by CAR-expressing T cells for treatment of MM. Clin Cancer Res; 20(15); 3989–4000. ©2014 AACR.