Retinoblastoma Protein-interacting Zinc Finger Gene Research Articles

Emerging Roles of PRDM Factors in Stem Cells and Neuronal System: Cofactor Dependent Regulation of PRDM3/16 and FOG1/2 (Novel PRDM Factors).

PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1) (PR) homologous domain containing (PRDM) transcription factors are expressed in neuronal and stem cell systems, and they exert multiple functions in a spatiotemporal manner. Therefore, it is believed that PRDM factors cooperate with a number of protein partners to regulate a critical set of genes required for maintenance of stem cell self-renewal and differentiation through genetic and epigenetic mechanisms. In this review, we summarize recent findings about the expression of PRDM factors and function in stem cell and neuronal systems with a focus on cofactor-dependent regulation of PRDM3/16 and FOG1/2. We put special attention on summarizing the effects of the PRDM proteins interaction with chromatin modulators (NuRD complex and CtBPs) on the stem cell characteristic and neuronal differentiation. Although PRDM factors are known to possess intrinsic enzyme activity, our literature analysis suggests that cofactor-dependent regulation of PRDM3/16 and FOG1/2 is also one of the important mechanisms to orchestrate bidirectional target gene regulation. Therefore, determining stem cell and neuronal-specific cofactors will help better understanding of PRDM3/16 and FOG1/2-controlled stem cell maintenance and neuronal differentiation. Finally, we discuss the clinical aspect of these PRDM factors in different diseases including cancer. Overall, this review will help further sharpen our knowledge of the function of the PRDM3/16 and FOG1/2 with hopes to open new research fields related to these factors in stem cell biology and neuroscience.

Deletion of the Prdm3 Gene Causes a Neuronal Differentiation Deficiency in P19 Cells.

PRDM (PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1) homologous domain-containing) transcription factors are a group of proteins that have a significant impact on organ development. In our study, we assessed the role of Prdm3 in neurogenesis and the mechanisms regulating its expression. We found that Prdm3 mRNA expression was induced during neurogenesis and that Prdm3 gene knockout caused premature neuronal differentiation of the P19 cells and enhanced the growth of non-neuronal cells. Interestingly, we found that Gata6 expression was also significantly upregulated during neurogenesis. We further studied the regulatory mechanism of Prdm3 expression. To determine the role of GATA6 in the regulation of Prdm3 mRNA expression, we used a luciferase-based reporter assay and found that Gata6 overexpression significantly increased the activity of the Prdm3 promoter. Finally, the combination of retinoic acid receptors α and β, along with Gata6 overexpression, further increased the activity of the luciferase reporter. Taken together, our results suggest that in the P19 cells, PRDM3 contributed to neurogenesis and its expression was stimulated by the synergism between GATA6 and the retinoic acid signaling pathway.

Multifaceted Role of PRDM Proteins in Human Cancer.

The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [Su(var)3-9, enhancer-of-zeste and trithorax] domain of histone methyltransferases (HMTs). These genes are involved in epigenetic regulation of gene expression through their intrinsic HMTase activity or via interactions with other chromatin modifying enzymes. In this way they control a broad spectrum of biological processes, including proliferation and differentiation control, cell cycle progression, and maintenance of immune cell homeostasis. In cancer, tumor-specific dysfunctions of PRDM genes alter their expression by genetic and/or epigenetic modifications. A common characteristic of most PRDM genes is to encode for two main molecular variants with or without the PR domain. They are generated by either alternative splicing or alternative use of different promoters and play opposite roles, particularly in cancer where their imbalance can be often observed. In this scenario, PRDM proteins are involved in cancer onset, invasion, and metastasis and their altered expression is related to poor prognosis and clinical outcome. These functions strongly suggest their potential use in cancer management as diagnostic or prognostic tools and as new targets of therapeutic intervention.

Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients.

Background: Retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) is a tumor suppressor deregulated in several human cancers. We aim to (1) explore RIZ1 expression in FIGO stages I–II cervical cancer tissues and its association with the clinical outcome of cervical cancer patients, (2) the role of RIZ1 in proliferation, apoptosis, migration, and invasion in cervical cancer cells.Methods: The expression of RIZ1 in 268 cervical cancer tissues and 30 paired adjacent non-tumor tissues were assessed by immunohistochemistry. We also examined RIZ1 at mRNA and protein level in 20 paired fresh frozen cervical cancer tissues and the adjacent non-tumor tissue using real-time PCR and western blot. We then examined proliferation, apoptosis, migration, and invasion in two human cervical cancer cells, HeLa and SiHa, with overexpression of RIZ1.Results: RIZ1 expression generally decreased in cervical cancer tissues. Decreased RIZ1 expression was significantly correlated with advanced FIGO stage (P = 0.005), deep stromal invasion (P = 0.001), lymphovascular space involvement (P = 0.041), pelvic lymph node metastasis (P = 0.005), and postoperative recurrence (P = 0.002). Kaplan-Meier analysis demonstrated that patients with low RIZ1 expression had shorter overall survival (OS) and disease-free survival (DFS) than those with high RIZ1 expression. Multivariate analysis showed that RIZ1 was an independent prognostic factor for DFS (HR = 2.184, 95% CI 1.365–3.496, P = 0.001) and OS (HR = 1.899, 95% CI 1.112–3.241, P = 0.019). In vitro analysis demonstrated that overexpression of RIZ1 inhibited cell proliferation, migration, and invasion, but promoted apoptosis in HeLa and SiHa cells.Conclusion: Down-regulation of RIZ1 may contribute to tumor migration, invasiveness, and poor survival of cervical cancer patients. RIZ1 may be a prognostic biomarker for cervical cancer patients.

PRDM14 promotes malignant phenotype and correlates with poor prognosis in colorectal cancer.

Emerging evidence suggests that stemness in cancer cells is a cause of drug resistance or metastasis and is an important therapeutic target. PR [positive regulatory domain I-binding factor 1 (PRDI-BF1) and retinoblastoma protein-interacting zinc finger gene (RIZ1)] domain containing 14 (PRDM14), that regulates pluripotency in primordial germ cell, has reported the overexpression and function of stemness in various malignancies, suggesting it as the possible therapeutic target. However, to our knowledge, there have been no reports on the expression and function of PRDM14 in colorectal cancer (CRC). Therefore, we investigated the expression and the role of PRDM14 in CRC. We performed immunohistochemistry evaluations and assessed PRDM14 expression on 414 primary CRC specimens. Colon cancer cell lines were subjected to functional and stemness assays in vitro and in vivo. We found that PRDM14 positive staining exhibited heterogeneity in the CRC primary tumor, especially at the tumor invasion front. The aberrant expression of PRDM14 at the invasion front was associated with lymph node metastasis and disease stage in patients with CRC. Furthermore, the multivariate analysis revealed high PRDM14 expression as an independent prognostic factor in the patients with Stage III CRC. Overexpression of PRDM14 enhanced the invasive, drug-resistant and stem-like properties in colon cancer cells in vitro and tumorigenicity in vivo. Our findings suggest that PRDM14 is involved in progression and chemoresistance of CRC, and is a potential prognostic biomarker and therapeutic target in the CRC patients.

Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated with Poor Survival and Aggressiveness of Cervical Cancer Patients

Background: Retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) is a tumor suppressor deregulated in several human cancers. We aimed to explore RIZ1 expression status in FIGO stages I–II cervical cancer tissues, the association of RIZ1 expression with the clinical outcome of cervical cancer patients, and the role of RIZ1 in the proliferation, apoptosis, migration, and invasion of cervical cancer cells. Methods: The expression of RIZ1 in 268 cervical cancer tissues was assessed by immunohistochemistry, real-time PCR, and immunoblotting. RIZ1 was ectopically overexpressed in HeLa and SiHa cells. MTT assay, Annexin V staining, flow cytometry, wound healing assay, and Transwell assay were used to assess cell proliferation, apoptosis, migration, and invasion. Results: RIZ1 expression generally decreased in cervical cancer tissues. Decreased RIZ1 expression was significantly correlated with advanced FIGO stage (P=0.005), deep stromal invasion (P=0.001), lymphovascular space involvement (P=0.041), pelvic lymph node metastasis (P=0.005), and postoperative recurrence (P=0.002). Kaplan–Meier analysis demonstrated that patients with low RIZ1 expression had shorter overall survival and disease-free survival (DFS) than those with high RIZ1 expression. Multivariate analysis showed that RIZ1 was an independent prognostic factor for DFS (HR=2.184 (95%CI,1.365-3.496), p=0.001) and OS (HR=1.899 (95%CI,1.112-3.241), p=0.019) of cervical cancer patients. Ectopic overexpression of RIZ1 inhibited the proliferation, migration, and invasion, but promoted the apoptosis of HeLa and SiHa cells in vitro. Conclusion: Downregulation of RIZ1 expression may contribute to the tumor progression and poor survival of cervical cancer patients. RIZ1 may be a prognostic biomarker for cervical cancer patients. Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81672584, 81602664, 81872460, 81372786), Natural Science Foundation of Heilongjiang Province (ZD2016016), Research project grant of Harbin Medical University Cancer Hospital (JY2016-05). Declaration of Interest: All the authors declare that there are no conflicts of interest. Ethical Approval: This study complied with the Helsinki Declaration and was approved by the Ethics Committee of Harbin Medical University (Harbin, China). Informed consents were obtained from all patients or their family members.

Mutations in A((8)) and A((9)) loci of exon 8 of retinoblastoma protein-interacting zinc finger gene of keloid patients

Objective: To study the situation of the mutations in the A((8)) and A((9)) loci of exon 8 of retinoblastoma protein-interacting zinc finger gene (RIZ) of keloid patients. Methods: From January 2003 to December 2007, 19 outpatient and hospitalized keloid patients of our hospital were conforming to the inclusion criteria. Both 3-5 g keloid tissue and 3 mL peripheral venous blood were collected from each patient to extract their genomic DNA, and the concentration was determined. The A((8)) and A((9)) loci fragments of exon 8 of RIZ were amplified by polymerase chain reaction (PCR). The length of product was detected by agarose gel electrophoresis, and DNA sequencing was performed after column chromatography. The mutations of A((8)) and A((9)) loci fragments were searched, and the types of mutations were determined. The consistency of genetic mutations of the keloid tissue and peripheral venous blood were compared. Data were processed with McNemar test. Results: The DNA concentrations of the extracted keloid tissue and peripheral venous blood were 0.54 and 0.37 μg/μL, respectively, which were above 0.10 μg/μL. The lengths of PCR products of A((8)) locus fragment DNA of exon 8 of RIZ from keloid tissue and peripheral venous blood were 235 and 238 bp, respectively, and those of A((9)) locus were 242 and 244 bp, respectively, which were basically the same as the designed DNA fragments. PCR products purity of A((8)) locus fragment DNA of exon 8 of RIZ from keloid tissue and peripheral venous blood were 1.81 and 1.75, respectively, and those of A((9)) locus were 1.82 and 1.78, respectively, which were above 1.50. Mutations in the A((8)) locus of exon 8 of RIZ were observed in keloid tissue of 18 patients, totally 6 gene mutations, including 4 point mutations and 2 frameshift mutations. Mutations in the A((9)) locus of exon 8 of RIZ were observed in keloid tissue of 9 patients, totally 9 gene mutations, including 7 point mutations and 2 frameshift mutations. No patient had a mutation in the A((8)) or A((9)) locus of exon 8 of RIZ in peripheral venous blood. Compared with those of peripheral venous blood, the mutations in the A((8)) and A((9)) loci of exon 8 of RIZ in keloid tissue of patients were statistically significant (χ(2)=16.06, 7.11, P<0.05). Conclusions: Point mutations and frameshift mutations occur in the A((8)) and A((9)) loci of exon 8 of RIZ in keloids of patients, which may be associated with the occurrence of keloids.

Combination of RIZ1 Overexpression and Radiotherapy Contributes to Apoptosis and DNA Damage of HeLa and SiHa Cervical Cancer Cells.

Although radiotherapy has been widely applied to treating cervical cancer in the clinic, its therapeutic efficacy is often restricted to the radioresistance of cancer cells. Retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) has been suggested as a tumour suppressor gene, whereas its role in cervical cancer with or without radiotherapy has been unclear. In this study, two cervical cancer cell lines, HeLa and SiHa cells, stably transfected with RIZ1 overexpression plasmid were subjected to ionizing radiation, and their survival fractions were calculated by assessing their clonogenic abilities. Our results showed that the forced overexpression of RIZ1 significantly reduced the clonogenic survival rates of both HeLa and SiHa cells exposed to ionizing radiation. By analysing the cell apoptotic status, we found that the RIZ1-overexpressed cervical cancer cells under ionizing radiation were more vulnerable to damage, and more γ-H2AX foci were found in these cells. Furthermore, the volumes of tumour xenografts formed by the RIZ1-overexpressed cells in nude mice under ionizing radiation were smaller than those generated by the control cells. There were more morphological changes, apoptosis cells and lower expression of PCNA in RIZ1-overexpressed tumour tissues of mice after exposure to ionizing radiation. Taken together, our study demonstrates that the overexpression of RIZ1 combined with radiotherapy facilitates apoptosis and DNA damage of cervical cancer cells.

Methylation Status of the RIZ1 Gene Promoter in Human Glioma Tissues and Cell Lines.

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1), a strong tumor suppressor, is silenced in many human cancers. Our previous studies showed that RIZ1 expression was negatively correlated with the grade of glioma and was a key predictor of patient survival. Therefore, RIZ1 could be a potential tumor suppressor during glioma pathogenesis, although the mechanism underlying RIZ1 gene inactivation in gliomas is unknown. We investigated the methylation status of the RIZ1 promoter in human glioma tissues and four glioblastoma (GBM) cell lines, and verified the effect of the methyltransferase inhibitor 5-aza-2-deoxycytidine (5-aza-CdR) on RIZ1 transcription and cell proliferation. Methylation-specific PCR (MSP) was performed to determine RIZ1 promoter methylation in human glioma specimens. The correlation between RIZ1 hypermethylation in tumors and clinicopathological features also was analyzed. 5-Aza-CdR treatment was used to reactivate gene expression silenced by hypermethylation in the U87 glioblastoma cell line, and real-time PCR was then used to measure RIZ1 expression. The ability of 5-aza-CdR to inhibit the proliferation of glioma cell lines whose RIZ1 promoters were hypermethylated was measured by bromodeoxyuridine (BrdU) incorporation. Among 51 human glioma specimens, RIZ1 promoter methylation was detected in 23 cases. Clinicopathological evaluation suggested that RIZ1 hypermethylation was negatively associated with tumor grade and patient age (P<0.05). Hypermethylation of the RIZ1 promoter was detected in the U87 and U251 cell lines. RIZ1 mRNA expression in U87 cells was upregulated after treatment with 5-aza-Cdr, which correlated with inhibition of cell proliferation in a time- and concentration-dependent manner. Promoter hypermethylation may play an important role in the epigenetic silencing of RIZ1 expression in human glioma tissues and GBM cell lines.

RIZ1: a potential tumor suppressor in glioma.

BackgroundRetinoblastoma protein-interacting zinc-finger gene 1 (RIZ1) displays strong tumor suppressive activities, and its expression is often silenced in many types of human tumors. However, the relationship between RIZ1 expression and glioma prognosis remains unclear.MethodsThe dysregulation of RIZ1 was evaluated using real-time polymerase chain reaction, western blot, and immunohistochemical analysis of gliomas from 51 patients. Correlation analysis was performed to examine relationships between RIZ1 immunoreactivity, clinicopathological features, and patient prognosis. Also, human malignant glioma U87 and U251 cell lines were stably transduced with ectogenic RIZ1 using a lentiviral vector to investigate the effects of induced expression of RIZ1 on cell proliferation, cell cycle, and apoptosis.ResultsReal-time polymerase chain reaction and western blot analysis showed that RIZ1 was downregulated in high-grade gliomas compared with low-grade gliomas and normal brain tissue. Immunohistochemistry showed less RIZ1 labeling in high-grade gliomas than in low-grade gliomas. There was a negative correlation between RIZ1 and Ki-67 immunoreactivity. Clinicopathological evaluation revealed that RIZ1 expression was negatively correlated with tumor grade and patient age. Kaplan-Meier survival analysis showed a positive correlation between RIZ1 immunoreactivity level and progression-free and overall survival times. Multivariate analysis showed that high RIZ1 expression was an independent prognostic factor for patients with gliomas. Induced expression of RIZ1 in U87 and U251 cells reduced cell proliferation and increased apoptosis, and cell cycle analysis revealed that a majority of cells were arrested at G2-M. Moreover, transfection with a RIZ1 expression vector increased p53 and caspase-3 expression and decreased p-IKBα and p-AKT protein levels, suggesting that RIZ1 may stimulate p53-mediated apoptosis and inhibit p-IKBα and p-AKT signaling pathways.ConclusionsOur results suggest that high RIZ1 labeling is indicative of lower grades of gliomas and is associated with better progression-free and overall survival rates. Therefore, RIZ1 may be a promising therapeutic target for patients with gliomas.

Aberrant Methylation of the 1p36 Tumor Suppressor Gene RIZ1 in Renal Cell Carcinoma.

Retinoblastoma protein-interacting zinc finger gene 1(RIZ1) functions as a tumor suppressor. Hypermethylation-mediated RIZ1 silencing has been reported in several cancers, but not in renal cell carcinoma (RCC) yet. We examined the RIZ1 expression and methylation in a panel of RCC cell lines and 50 primary tumors using semiquantitative/quantitative polymerase chain reaction (PCR), methylation specific PCR, and bisulfite sequencing genomic. We also explored the relationship between methylation status of RIZ1 and clinicopathological features in RCC patients. RIZ1 expression was down-regulated or lost in OS-RC-2, 769-P, Caki-1, 786-O and A498 RCC cell lines. Restored expression of RIZ1 was detected after addition of 5-aza-2'-deoxycytidine with/without trichostatin A, suggesting that DNA methylation directly mediates its silencing. The RIZ1 expression was significantly reduced in RCCs compared to adjacent non-malignant renal samples (P<0.001). Aberrant methylation was detected in 15 of 50 (30%) RCCs and in 2 of 28 (7%) adjacent non- malignant renal samples (P=0.02). No statistically significant correlation between methylated and unmethylated cases with regard to age, gender, pathological stage and grade was observed. RIZ1 expression is down-regulated in human RCC, and this down-regulation is associated with methylation. RIZ1 methylation may play a role in renal carcinogenesis.

PRDI-BF1-RIZ domain of retinoblastoma protein-interacting zinc finger gene 1 induces apoptosis and exerts anticancer activity in esophageal squamous cell carcinoma cells.

The present study examined the role of the PRDI-BF1-RIZ (PR) domain of tumor suppressor retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) as an anticancer domain and its ability to induce apoptosis in esophageal squamous cell carcinoma (ESCC) cells. The TE13 ESCC cell line was transfected with pcDNA3.1(+) eukaryotic expression vectors bearing the open reading frames of either the human RIZ1 gene or the PR domain, and the mRNA and protein expression levels were then detected using quantitative reverse transcription polymerase chain reaction and western blotting, respectively. The rate of apoptosis was determined by flow cytometry and the cell invasion ability was determined by an invasion assay. RIZ1 and the PR domain induced apoptosis and reduced the cell invasion ability (P<0.01). These findings indicate that the RIZ1 gene possesses anticancer activity in the PR domain, which may be important in inhibiting the development of ESCC.

Tumor Suppressor RIZ1 in Carcinogenesis

Human retinoblastoma protein-interacting zinc-finger gene RIZ (PRDM2) encodes two protein products, tumor suppressor RIZ1 and proto-oncoprotein RIZ2, using alternative promoters. RIZ1 and RIZ2 regulate normal cell division in a Yin-Yang fashion with RIZ1 arresting cells in G2/M phase and inducing apoptosis and RIZ2 promoting cell proliferation. Silenced RIZ1 expression has been detected in various types of cancer. Because both RIZ isoforms contain multiple functional domains, their function mechanisms in suppressing or promoting tumor growth are complex. Based on the current knowledge, it is rational to propose four potential routes for RIZ1 to exert its tumor suppressing functions: directly repressing the promoters of growth factors such as insulin-like growth factor-1 via H3K9 (histone H3 lysine 9) methylation, regulating estrogen-induced pS2 transcription through forming a complex with transcriptional co-activator p300, activating tumor suppressor p53 using a methylation-acetylation interplay, and blocking gene transcriptions by binding to PR-Set7 and establishing a H4K20me1 (histone H4 lysine 20 monomethylation) - H3K9me1 (histone H3 lysine 9 mono-methylation) trans-tail ‘histone code’ at an ectopic locus.

Change of the expression profile in esophageal carcinoma cells following up-regulated retinoblastoma protein-interacting zinc finger gene 1 gene

Objective To observe the effects of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) gene on the expression profile of esophageal squamous cell carcinoma cell line TE13.Methods The TE13 cells were cultured and divided into 10 portions,subcultured and transfected respectively by pcDNA3.1 (+) and pcDNA3.1 (+)/RIZ1 using the lipofectamin transfection reagent.After successful transfection,Agilent whole human genome oligo microarray (4 * 44K) was used to screen the effects of RIZ1 on the expression profile of esophageal carcinoma cell line TE13 (foldchange ≥2 or ≤0.5).The test results was confirmed by real-time quantitative polymerase chain reaction (real-time PCR).Results The DNA microarray data suggested that RIZ1 transfection induced widespread change of gene expression profile of TE13 cells.The up-regulated and down-regulated genes were amount to 960 and 1 163 respectively.The altered genes belonged to many functional categories,mainly in cell development,regulation of viral reproduction.RIZ1 gene may be involved in some pathways,such as cytokine receptor interaction,TGF-beta signaling pathway.Conclusion Widespread change of gene expression profile occurred after transfection with RIZ1 gene.We postulate that RIZ1 inhibits the tumor via the effect of these genes and involvement in pathways.The study provides the evidence for elucidating the relation between RIZ1 gene and the development and progress of esophageal cancer. Key words: Esophageal squamous cell carcinoma; Retinoblastoma protein-interacting zinc finger gene 1; DNA microarray

The role of the retinoblastoma protein-interacting zinc finger gene 1 tumor suppressor gene in human esophageal squamous cell carcinoma cells.

The tumor suppressor protein retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) is downregulated in several types of cancer, including esophageal squamous cell carcinoma (ESCC). The present study used two in vitro methods to re-express RIZ1 in the human ESCC TE13 cell line in order to induce apoptosis. RIZ1 was re-expressed in the TE13 cells by reintroducing the gene through transfection or by removal of transcriptional repression through treatment with a DNA methyltransferase (DNMT) inhibitor. To reintroduce the gene, the open reading frame of the RIZ1 gene was inserted into the eukaryotic expression pcDNA3.1(+) vector and pcDNA3.1(+)/RIZ1 was purified and transfected into the TE13 ESCC cells. Removing transcriptional repression involved treating the TE13 cells with 5-aza-2′-deoxycytidine (5-aza-CdR), a DNMT inhibitor. RIZ1 mRNA and protein expression were determined by quantitative polymerase chain reaction (qPCR) and western blotting. The rate of apoptosis of the cells was determined by flow cytometry. A recombinant eukaryotic human RIZ1 expression plasmid, pcDNA3.1(+)/RIZ1, was constructed and confirmed by sequencing. RIZ1 mRNA and protein expression increased in pcDNA3.1(+)/RIZ1 stably transfected cells. Treatment with 5-aza-CdR for 48 and 72 h resulted in increased RIZ1 protein expression and increased the rate of apoptosis in the TE13 cells (P<0.01). In conclusion, transfection of the TE13 cells with the eukaryotic pcDNA3.1(+)/RIZ1 expression vector and reversal of transcriptional repression of RIZ1 using 5-aza-CdR demonstrate that it is possible to re-express RIZ1 in TE13 cells. Furthermore, the re-expression of RIZ1 led to an increased rate of apoptosis and this method may provide new therapeutic possibilities.

Alteration in gene expression profile and oncogenicity of esophageal squamous cell carcinoma byRIZ1upregulation

To investigate the effect of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) upregulation in gene expression profile and oncogenicity of human esophageal squamous cell carcinoma (ESCC) cell line TE13. TE13 cells were transfected with pcDNA3.1(+)/RIZ1 and pcDNA3.1(+). Changes in gene expression profile were screened and the microarray results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Nude mice were inoculated with TE13 cells to establish ESCC xenografts. After two weeks, the inoculated mice were randomly divided into three groups. Tumors were injected with normal saline, transfection reagent pcDNA3.1(+) and transfection reagent pcDNA3.1(+)/RIZ1, respectively. Tumor development was quantified, and changes in gene expression of RIZ1 transfected tumors were detected by RT-PCR and Western blotting. DNA microarray data showed that RIZ1 transfection induced widespread changes in gene expression profile of cell line TE13, with 960 genes upregulated and 1163 downregulated. Treatment of tumor xenografts with RIZ1 recombinant plasmid significantly inhibited tumor growth, decreased tumor size, and increased expression of RIZ1 mRNA compared to control groups. The changes in gene expression profile were also observed in vivo after RIZ1 transfection. Most of the differentially expressed genes were associated with cell development, supervision of viral replication, lymphocyte costimulatory and immune system development in esophageal cells. RIZ1 gene may be involved in multiple cancer pathways, such as cytokine receptor interaction and transforming growth factor beta signaling. The development and progression of esophageal cancer are related to the inactivation of RIZ1. Virus infection may also be an important factor.

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1) dysregulation in human malignant meningiomas

Retinoblastoma protein-interacting zinc-finger gene 1 (RIZ1) expression is often silenced in many types of human tumors. However, the relationship between RIZ1 expression and malignant meningiomas remains unclear. Here we have found for the first time that the expression of RIZ1 genes are associated with meningiomas progression through extensive analyses of Affymetrix GeneChip microarray data. Further validation methods for gene expression included quantitative PCR (qPCR), western blot and immunohistochemistry analysis, and these methods confirmed that RIZ1 is significantly downregulated in malignant meningioma tissues, as compared with benign meningiomas. In addition, malignant meningioma cells were stably transfected with ectogenic RIZ1 using Lentivirus-mediated transfection, and the transfections were followed by an in vitro 5-bromo-2-deoxyuridin incorporation assay, colony formation assay, cell cycle analysis, invasive analysis, apoptotic assay and western blot analysis. Our results demonstrate that the forced expression of RIZ1 in a malignant meningioma cell line inhibited cellular proliferation and arrested the cells in the G2/M phase of the cell cycle. We also confirmed that overexpression of RIZ1 may induce apoptosis of malignant meningioma cells. Furthermore, RIZ1 overexpression in malignant meningioma cells was associated with the downregulation of c-myc expression. These results from our study indicate that RIZ1 expression is significantly downregulated as the formation of meningiomas progressed, and suggest that RIZ1 may represent a promising candidate tumor suppressor gene that contributes to malignant meningiomas.

Study onRIZ1gene promoter methylation status in human esophageal squamous cell carcinoma

To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) in the human esophageal squamous cell carcinoma (ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogenesis, tumor progression and metastasis etc of ESCC. Methylation-specific polymerase chain reaction (MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was detected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozen pathological specimens from 47 ESCC patients were performed using the same MSP methodology. Promoter methylation of RIZ1 gene was detected in TE13, CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study. The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statistically significant (χ(2) = 24.136, P < 0.01). Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant. Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.

Identification of a functional estrogen‐responsive enhancer element in the promoter 2 of PRDM2 gene in breast cancer cell lines

The retinoblastoma protein-interacting zinc-finger (RIZ) gene, also known as PRDM2, encodes two protein products, RIZ1 and RIZ2, differing for the presence of a 202 aa domain, called PR domain, at the N-terminus of the RIZ1 molecule. While the histone H3 K9 methyltransferase activity of RIZ1 is associated with the negative control of cell proliferation, no information is currently available on either expression regulation of the RIZ2 form or on its biological activity. RIZ proteins act as ER co-activators and promote optimal estrogen response in female reproductive tissues. In estrogen-responsive cells, 17-β estradiol modulates RIZ gene expression producing a shift in the balanced expression of the two forms. Here, we demonstrate that an estrogen-responsive element (ERE) within the RIZ promoter 2 is regulated in a ligand-specific manner by ERα, through both the AF1 and AF2 domains. The pattern of ERα binding, histone H4 acetylation, and histone H3 cyclical methylation of lysine 9 was comparable to other estrogen-regulated promoters. Association of topoisomerase IIβ with the RIZ promoter 2 confirmed the transcriptional activation induced by estrogen. We hypothesize that RIZ2, acting as a negative regulator of RIZ1 function, mediates the proliferative effect of estrogen through regulation of survival and differentiation gene expression.

Methylation of the RIZ1 Gene In Myelodysplastic Syndrome and Acute Myeloid Leukemia

AbstractAbstract 5112Inactivation of a tumor suppressor gene is often caused by a mutation, small deletion of one allele accompanied by loss of the second allele. Methylation in a promoter CpG of several tumor suppressor genes has recently been reported and has been associated with loss or decreased expression in many tumors. We previously reported frequent loss of heterozygosity on the short arm of chromosome 1 (1p) in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). The retinoblastoma protein-interacting zinc finger gene RIZ maps to 1p36. Mouse gene knockout models show that RIZ1 inactivation can cause tumor susceptibility. Inactivation of the RIZ1 gene by promoter hypermethylation has been reported in breast, liver, and gastric carcinoma. Previous study showed altered expression of the RIZ1 gene in human leukemia. However, methylation status of the RIZ1 gene has not been well studied in hematological neoplasms. To determine the relevance of the RIZ1 methylation, we performed methylation specific-polymerase chain reaction (PCR) analysis on the RIZ1 gene in 34 patients with MDS and 17 with AML evolved from MDS (secondary AML) as well as 55 patients with de novo AML. The 34 MDS samples consisted of 13 refractory anemia (RA), 1 RA with ringed sideroblasts (RARS), 10 RA with excess of blasts (RAEB), 6 RAEB in transformation (RAEB-t), and 4 chronic myelomonocytic leukemia. The 55 de novo AML consisted of 1 M0, 12 M1, 17 M2, 7 M3, 8 M4, 7 M5, 1 M6, and 2 M7. Written informed consent was obtained from the patients. Methylation of the RIZ1 gene was detected in 17 of the 34 MDS (50%) and 22 of 72 de novo and secondary AML (31%) (p=0.053). Methylation was detected in 7 of 14 low risk MDS (50%) and 10 of 20 high risk MDS (50%). Patients with MDS were classified using the IPSS score. Frequency of methylation was not statistically different among IPSS subgroups (p=0.419). No statistical differences were observed between methylation and overall survival (3 years) or progression to AML. In AML patients, methylation was more frequent in secondary AML (11/17, 65%) than in de novo AML (11/55, 20%) (p=0.0005). To define the methylation status of the CpG in the RIZ1 promoter region, we performed bisulfite sequence in several samples with methylation. Bisulfite sequence analysis revealed methylation at many CpG sites in the promoter region. Expression of the RIZ1 gene was examined by quantitative real time reverse transcriptase-PCR analysis in 22 samples of MDS and AML. RIZ1 expression (mean) was not statistically different in secondary AML and de novo AML (2.026 vs. 1.900, p=0.815). RIZ1 expression (mean) was not statistically different in methylation-positive group and methylation-negative group (1.996 vs. 1.810, p=0.728). In comparison with expression of normal bone marrow cells, decreased RIZ1 expression was accompanied by methylation in 6 of 9 samples examined, while it was also observed in 7 of 13 without methylation. HL-60 myeloid leukemia cells with RIZ1 methylation were cultivated for 3 days in the presence of various concentrations of 5-Aza-dC. Treatment of the leukemia cells with 5 Aza-dC induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene was inactivated in MDS and AML in part by methylation, whereas another mechanism of inactivation should be involved in others. Disclosures:No relevant conflicts of interest to declare.