Topotecan and methotrexate alter expression of the apoptosis-related genes BCL2, FAS and BCL2L12 in leukemic HL-60 cells

The BCL2 family of genes (B-cell CLL/lymphoma 2; Bcl-2) plays a pivotal role in the highly regulated process of apoptosis. We have recently cloned a newly identified member of this family, BCL2L12, which was found to be differentially expressed in many tumors. It is known that topotecan and methotrexate act through induction of apoptosis in cancer cells. In the present study we investigated the expression profile of the novel apoptotic gene BCL2L12 in relation to other apoptotic genes in the human leukemic cell line HL-60, after treatment with topotecan or methotrexate. The kinetics of apoptosis induction and cell toxicity were investigated by DNA laddering and the MTT method, respectively. Gene expression levels were analyzed by RT-PCR using gene-specific primers. Downregulation of BCL2L12, BCL2 and FAS was observed after treatment of HL-60 cells with topotecan, while treatment with methotrexate led to downregulation of BCL2 and FAS, with no change in BCL2L12 expression. Our results support the significance of mRNA modulations in the expression of apoptosis-related genes during treatment of human leukemic cells with anticancer drugs.     

5-杂氮-2′-脱氧胞苷诱导鼻咽癌细胞株Syk基因启动子去甲基化的研究

利用5-杂氮-2′-脱氧胞苷（5-aza-2′-deoxycytidine,5-aza-CdR）处理体外培养的鼻咽癌细胞株CNE-1、CNE-2及永生化非癌性人鼻咽上皮细胞株NP-69,采用BS-PCR、Q-RT-PCR及Westernblot方法分别检测经5μmol/L的5-aza-CdR处理前后,各细胞株中Syk基因启动子甲基化状况及SykmRNA和蛋白质表达情况。探讨去甲基化药物5-杂氮-2′-脱氧胞苷（5-aza-CdR）对鼻咽癌细胞株中脾酪氨酸激酶（spleen tyrosine kinase,Syk）启动子甲基化水平及其表达的影响。结果显示,Syk基因启动子甲基化水平与鼻咽癌细胞分化程度呈负相关,两种鼻咽癌细胞株的Syk mRNA和蛋白质表达水平显著低于NP-69细胞（P〈0.01）;经5-aza-CdR处理后两种鼻咽癌细胞株的Syk基因启动子甲基化水平降低,Syk mRNA及蛋白质表达升高（P〈0.05）;高分化鼻咽癌细胞株对药物敏感性高于低分化鼻咽癌细胞株（P〈0.01）。由此可见,两种鼻咽癌细胞株中存在不同程度的Syk基因启动子甲基化状态,5-aza-CdR能有效逆转鼻咽癌细胞株Syk基因启动子的甲基化状态,升高Syk mRNA及蛋白质表达,同时鼻咽癌细胞分化程度越高恢复Syk基因表达的比率越高。     

Treatment of MCF-7 cells with taxol and etoposide induces distinct alterations in the expression of apoptosis-related genes BCL2, BCL2L12, BAX, CASPASE-9 and FAS

We studied alterations in the mRNA expression levels of BCL2 (Bcl-2), BCL2L12, BAX, FAS and CASPASE-9 genes in the MCF-7 breast cancer cell line in response to treatment with two anticancer drugs. Cell toxicity was evaluated by the MTT method, trypan blue staining and DNA laddering, whereas the expression levels of the apoptosis-related genes were analysed by RT-PCR using gene-specific primers. In the case of etoposide, down-regulation of the BCL2L12-A gene variant and of CASPASE-9, as well as upregulation of BAX, was observed, whereas treatment of MCF-7 cells with taxol led to down-regulation of the mRNA levels of all genes examined. Our results support the idea that after long-term clinical studies, mRNA expression analysis of BCL2L12 and other members of the BCL2 gene family may serve as useful molecular markers predicting chemotherapy response in breast cancer.     

l-DOPA Decarboxylase (DDC) Expression Status as a Novel Molecular Tumor Marker for Diagnostic and Prognostic Purposes in Laryngeal Cancer

l-DOPA decarboxylase (DDC) plays an essential role in the enzymatic synthesis of dopamine and alterations in its gene expression have been reported in several malignancies. Our objective was to analyze DDC messenger RNA (mRNA) and protein expression in laryngeal tissues and to evaluate the clinical implication of this molecule in laryngeal cancer. In this study, total RNA was isolated from 157 tissue samples surgically removed from 100 laryngeal cancer patients. A highly sensitive real-time polymerase chain reaction methodology based on SYBR Green I fluorescent dye was developed for the quantification of DDC mRNA levels. In addition, Western blot analysis was performed for the detection of DDC protein. DDC mRNA expression was revealed to be significantly downregulated in primary laryngeal cancer samples compared with their nonmalignant counterparts (P = .001). A significant negative association was also disclosed between DDC mRNA levels and TNM staging (P = .034). Univariate analysis showed that patients bearing DDC-positive tumors had a significantly decreased risk of death (hazard ratio = 0.23, P = .012) and local recurrence (hazard ratio = 0.32, P =.006), whereas DDC expression retained its favorable prognostic significance in the multivariate analysis. Kaplan-Meier curves further demonstrated that DDC-positive patients experienced longer overall and disease-free survival periods (P = .006 and P = .004, respectively). Moreover, DDC protein was detected in both neoplastic and noncancerous tissues. Therefore, our results suggest that DDC expression status could qualify as a promising biomarker for the future clinical management of laryngeal cancer patients.     

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.     

Evaluation of the prognostic value of TGF-β superfamily type I receptor and TGF-β type II receptor expression in nasopharyngeal carcinoma using high-throughput tissue microarrays

Gene expression profiling had revealed that TGF-β superfamily type I receptor (also known as activin receptor-like kinase-1, ALK1) and TGFβR2 (TGF-β type II receptor) were down-regulated in nasopharyngeal carcinoma (NPC) (P < 0.05, respectively). However, no study with significantly large clinical samples to address the relevance of ALK1 and TGFβR2 in NPC progression or in patient outcomes has been reported. This study aims to assess the possible correlations of ALK1 and TGFβR2 expression with NPC progression and their potential prognostic predictive ability in NPC outcomes. ALK1 and TGFβR2 mRNA and protein levels were detected by qRT-PCR and NPC tissue microarray (TMA), which included 742 tissue cores. Both mRNA and protein levels of ALK1 and TGFβR2 were significantly lower in the cancer tissues compared with the non-cancerous tissues (P < 0.05). Epstein-Barr virus small RNA (EBER-1) hybridization signals in NPC showed significant associations with ALK1 and TGFβR2 proteins (P = 0.000 and 0.003, respectively). In the final logistic regression analysis model, the abnormal expression of ALK1 and TGFβR2 were found to be independent contributors to nasopharyngeal carcinogenesis (P = 0.000 and 0.000, respectively). A survival analysis revealed that ALK1 (Disease Free Survival (DFS): P = 0.002, Overall Survival (OS): P = 0.007) and TGFβR2 (DFS: P = 0.072, OS: P = 0.045) could predict the prognosis of NPC patients. The positive expression of ALK1 and TGFβR2 were independent risk factors for DFS and OS in multivariate analyses (DFS: P = 0.001 and 0.420, respectively; OS: P = 0.018 and 0.047, respectively). These results suggest that ALK1 and TGFβR2 may be useful prognostic biomarkers in NPC.     

N6-methyladenosine modification of circCUX1 confers radioresistance of hypopharyngeal squamous cell carcinoma through caspase1 pathway

Hypopharyngeal squamous cell carcinoma (HPSCC) is one of the most common malignant tumors in otolaryngology head and neck surgery and is one of the worst prognostic malignant tumors. Endogenous circular RNA (circRNA) is more stable than mRNA, microRNA (miRNA), and long non-coding RNA (LncRNA) in exosomes, plasma, and urine, and participates in gene expression regulation to perform different functions. Therefore, circRNA is expected to become a biomarker and therapy target for many tumors. However, the expression and function of circRNA regulated by N6-methyladenosine (m6A) are still unclear in HNSCC. In this study, we demonstrated that a specific circRNA, circCUX1, was upregulated in HPSCC patients who are resistant to radiotherapy and predicts poor survival outcome. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circCUX1 and stabilizes its expression. Knockdown circCUX1 promotes the sensitivity of hypopharyngeal cancer cells to radiotherapy. In addition, circCUX1 binds to Caspase1 and inhibits its expression, resulting in a decrease in the release of inflammatory factors, thereby developing tolerance to radiotherapy. Our findings indicate that circCUX1 is a potential therapeutic target for radiotherapy tolerance in HPSCC patients.Subject terms: Radiotherapy, Head and neck cancer     

Identification of PRDX4 and P4HA2 as metastasis-associated proteins in oral cavity squamous cell carcinoma by comparative tissue proteomics of microdissected specimens using iTRAQ technology

Cervical lymph node metastasis represents the major prognosticator for oral cavity squamous cell carcinoma (OSCC). Here, we used an iTRAQ-based quantitative proteomic approach to identify proteins that are differentially expressed between microdissected primary and metastatic OSCC tumors. The selected candidates were examined in tissue sections via immunohistochemistry, and their roles in OSCC cell function investigated using RNA interference. Seventy-four differentially expressed proteins in nodal metastases, including PRDX4 and P4HA2, were identified. Immunohistochemical analysis revealed significantly higher levels of PRDX4 and P4HA2 in tumor cells than adjacent non-tumor epithelia (P < 0.0001 and P < 0.0001, respectively), and even higher expression in the 31 metastatic tumors of lymph nodes, compared to the corresponding primary tumors (P = 0.060 and P = 0.002, respectively). Overexpression of PRDX4 and P4HA2 was significantly associated with positive pN status (P = 0.048 and P = 0.021, respectively). PRDX4 overexpression was a significant prognostic factor for disease-specific survival in both univariate and multivariate analyses (P = 0.034 and P = 0.032, respectively). Additionally, cell migration and invasiveness were attenuated in OEC-M1 cells upon in vitro knockdown of PRDX4 and P4HA2 with specific interfering RNA. Novel metastasis-related prognostic markers for OSCC could be identified by our approach.     

长非编码RNA THOR在肿瘤中的作用

长非编码RNA（long non-coding RNA,lncRNA）是一类长度超过200 nt并且缺乏蛋白质编码潜能的RNA分子。最初lncRNA被认为是由RNA聚合酶Ⅱ转录的副产物,且无生物学功能。随着转录组测序技术的发展,大规模的lncRNA被鉴定出来。越来越多的证据表明,lncRNA参与多种生物学过程,包括基因印记、基因组重排、染色质修饰、细胞周期调控、转录、剪接、mRNA降解和翻译。lncRNA异常表达与人类多种疾病相关,尤其是增生性疾病,包括胃癌、肝癌和直肠癌等。其中,睾丸相关的高度保守的致癌长非编码RNA（testis-associated highly-conserved oncogenic long non-coding RNA,THOR）是一种非常保守的非编码RNA,在睾丸中特异性表达,并广泛存在于人的多种肿瘤组织中,如肝癌、胃癌、鼻咽癌、肾细胞癌、骨肉瘤、视网膜母细胞瘤、黑色素瘤、非小细胞肺癌和舌鳞状细胞癌中,在其发生和发展过程中发挥重要作用。在鼻咽癌、肾细胞癌、骨肉瘤、黑色素瘤、非小细胞肺癌和舌鳞状细胞癌中,THOR主要通过与胰岛素样生长因子2 mRNA结合蛋白1（insulin-like growth factor 2 mRNA-binding protein 1,IGF2BP1）相互作用,促进肿瘤细胞的增殖。在肝癌中,THOR分别通过PTEN/AKT和β-联蛋白信号促进癌细胞的增殖和肝肿瘤干细胞的扩增。在胃癌和骨肉瘤中,THOR主要通过提高SOX9的表达增强癌细胞的干性。在视网膜母细胞瘤中,THOR主要通过提高c-myc的表达促进癌细胞增殖。在鼻咽癌中,THOR主要通过提高YAP的表达增强癌细胞的干性。     

长非编码RNA THOR在肿瘤中的作用

长非编码RNA（long non-coding RNA,lncRNA）是一类长度超过200 nt并且缺乏蛋白质编码潜能的RNA分子。最初lncRNA被认为是由RNA聚合酶Ⅱ转录的副产物,且无生物学功能。随着转录组测序技术的发展,大规模的lncRNA被鉴定出来。越来越多的证据表明,lncRNA参与多种生物学过程,包括基因印记、基因组重排、染色质修饰、细胞周期调控、转录、剪接、mRNA降解和翻译。lncRNA异常表达与人类多种疾病相关,尤其是增生性疾病,包括胃癌、肝癌和直肠癌等。其中,睾丸相关的高度保守的致癌长非编码RNA（testis-associated highly-conserved oncogenic long non-coding RNA,THOR）是一种非常保守的非编码RNA,在睾丸中特异性表达,并广泛存在于人的多种肿瘤组织中,如肝癌、胃癌、鼻咽癌、肾细胞癌、骨肉瘤、视网膜母细胞瘤、黑色素瘤、非小细胞肺癌和舌鳞状细胞癌中,在其发生和发展过程中发挥重要作用。在鼻咽癌、肾细胞癌、骨肉瘤、黑色素瘤、非小细胞肺癌和舌鳞状细胞癌中,THOR主要通过与胰岛素样生长因子2 mRNA结合蛋白1（insulin-like growth factor 2 mRNA-binding protein 1,IGF2BP1）相互作用,促进肿瘤细胞的增殖。在肝癌中,THOR分别通过PTEN/AKT和β-联蛋白信号促进癌细胞的增殖和肝肿瘤干细胞的扩增。在胃癌和骨肉瘤中,THOR主要通过提高SOX9的表达增强癌细胞的干性。在视网膜母细胞瘤中,THOR主要通过提高c-myc的表达促进癌细胞增殖。在鼻咽癌中,THOR主要通过提高YAP的表达增强癌细胞的干性。     

BCL10 expression is unrelated to clinico-pathological parameters or prognoses for oral squamous cell carcinomas

BCL-10 (B-cell lymphoma 10) has been linked to a pro-apoptotic gene in mucosa-associated lymphoid tissue (MALT) lymphomas. We describe the expression of BCL10 in oral squamous cell carcinoma (OSCC) and its relation to clinical, pathological and prognostic parameters. We carried out a retrospective study of 50 patients in Spain who were diagnosed with OSCC. We constructed a tissue microarray of the samples to study the expression of BCL10 using immunohistochemistry. Diffuse and homogeneous staining was observed in the nuclei and cytoplasms of most neoplastic cells of the vast majority of tumors and no significant differences were seen in different areas of the tumors. The expression was unrelated to any clinical or pathological parameters including tumor stage. The intra-class coefficient was 0.97, which indicates the minimal variability among the determinations.     

BCL10 expression is unrelated to clinico-pathological parameters or prognoses for oral squamous cell carcinomas

Abstract

BCL-10 (B-cell lymphoma 10) has been linked to a pro-apoptotic gene in mucosa-associated lymphoid tissue (MALT) lymphomas. We describe the expression of BCL10 in oral squamous cell carcinoma (OSCC) and its relation to clinical, pathological and prognostic parameters. We carried out a retrospective study of 50 patients in Spain who were diagnosed with OSCC. We constructed a tissue microarray of the samples to study the expression of BCL10 using immunohistochemistry. Diffuse and homogeneous staining was observed in the nuclei and cytoplasms of most neoplastic cells of the vast majority of tumors and no significant differences were seen in different areas of the tumors. The expression was unrelated to any clinical or pathological parameters including tumor stage. The intra-class coefficient was 0.97, which indicates the minimal variability among the determinations.     

Linkage between STAT regulation and Epstein-Barr virus gene expression in tumors

Epstein-Barr virus (EBV) latency gene expression in lymphoblastoid cell lines is regulated by EBNA2. However, the factors regulating viral expression in EBV-associated tumors that do not express EBNA2 are poorly understood. In EBV-associated tumors, EBNA1 and frequently LMP1 are synthesized. We found that an alternative latent membrane protein 1 (LMP1) promoter, L1-TR, located within the terminal repeats is active in both nasopharyngeal carcinoma and Hodgkin's disease tissues. Examination of the L1-TR and the standard ED-L1 LMP1 promoters in electrophoretic mobility shift assays revealed that both promoters contain functional STAT binding sites. Further, both LMP1 promoters responded in reporter assays to activation of JAK-STAT signaling. Cotransfection of JAK1 or v-Src or treatment of cells with the cytokine interleukin-6 upregulated expression from ED-L1 and L1-TR reporter plasmids. Cotransfection of a dominant negative STAT3 beta revealed that STAT3 is likely to be the biologically relevant STAT for EBNA1 Qp and LMP1 L1-TR promoter regulation. In contrast, LMP1 expression from ED-L1 was not abrogated by STAT3 beta, indicating that the two LMP1 promoters are regulated by different STAT family members. Taken together with the previous demonstration of JAK-STAT activation of Qp driven EBNA1 expression, this places two of the EBV genes most commonly expressed in tumors under the control of the same signal transduction pathway. Immunohistochemical analyses of nasopharyngeal carcinoma tumors revealed that STAT3, STAT5, and STAT1 are constitutively activated in these tumors while STAT3 is constitutively activated in the malignant cells of Hodgkin's disease. We hypothesize that chronic or aberrant STAT activation may be both a necessary and predisposing event for EBV-driven tumorigenesis in immunocompetent individuals.