Expression Of RIZ1 Research Articles

Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

BackgroundT cell activation and programming from their naïve/resting state, characterized by widespread modifications in chromatin accessibility triggering extensive changes in transcriptional programs, is orchestrated by several cytokines and transcription regulators. PRDM1 and PRDM2 encode for proteins with PR/SET and zinc finger domains that control several biological processes, including cell differentiation, through epigenetic regulation of gene expression. Different transcripts leading to main protein isoforms with (PR +) or without (PR-) the PR/SET domain have been described. Although many studies have established the critical PRDM1 role in hematopoietic cell differentiation, maintenance and/or function, the single transcript contribution has not been investigated before. Otherwise, very few evidence is currently available on PRDM2. Here, we aimed to analyze the role of PRDM1 and PRDM2 different transcripts as mediators of T lymphocyte activation.MethodsWe analyzed the transcription signature of the main variants from PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) genes, in human T lymphocytes and Jurkat cells overexpressing PRDM2 cDNAs following activation through different signals.ResultsT lymphocyte activation induced an early increase of RIZ2 and RIZ1 followed by BLIMP1b increase and finally by BLIMP1a increase. The “first” and the “second” signals shifted the balance towards the PR- forms for both genes. Interestingly, the PI3K signaling pathway modulated the RIZ1/RIZ2 ratio in favor of RIZ1 while the balance versus RIZ2 was promoted by MAPK pathway. Cytokines mediating different Jak/Stat signaling pathways (third signal) early modulated the expression of PRDM1 and PRDM2 and the relationship of their different transcripts confirming the early increase of the PR- transcripts. Different responses of T cell subpopulations were also observed. Jurkat cells showed that the acute transient RIZ2 increase promoted the balancing of PRDM1 forms towards BLIMP1b. The stable forced expression of RIZ1 or RIZ2 induced a significant variation in the expression of key transcription factors involved in T lymphocyte differentiation. The BLIMP1a/b balance shifted in favor of BLIMP1a in RIZ1-overexpressing cells and of BLIMP1b in RIZ2-overexpressing cells.ConclusionsThis study provides the first characterization of PRDM2 in T-lymphocyte activation/differentiation and novel insights on PRDM1 and PRDM2 transcription regulation during initial activation phases.

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.

RIZ1 is regulated by estrogen and suppresses tumor progression in endometrial cancer

Endometrial cancer (EC) is the estrogen-dependent gynecologic malignancy, however the molecular mechanism involved in the development and progression of EC remain unclear. The aim of this study was to investigate the role of RIZ1 in EC. Immunohistochemical analysis revealed that RIZ1was decreased in EC than in normal endometrium. Lower RIZ1 level was correlated with high-grade carcinoma (p = 0.048) and positive expression of ERα (p = 0.004). In EC cells, estrogen could down regulated the expression of RIZ1, however, ICI182,780 could up regulated the expression of RIZ1. Besides, in vitro and in vivo, RIZ1 could remarkably suppress tumor proliferation, metastasis and invasion. Our data support that RIZ1 was a novel tumor suppressor and could provide a potential therapeutic target in human EC.

Epigenetics of Human Parathyroid Tumors

Parathyroid tumors are common endocrine neoplasia associated with primary hyperparathyroidism, a metabolic disorder sustained by parathormone hypersecretion. The epigenetic scenario in parathyroid tumors is beginning to be decoded. Here, main findings are reviewed: hypermethylation of specific DNA CpG islands has been described, despite global DNA promoter hypomethylation was not detectable; embryonic-related miRNAs, belonging to the C19MC and miR‐371-373 clusters, and miR‐296, are deregulated; expression of histone H1.2 and H2B is increased; expression of histone methyltransferase EZH2, BMI1 and RIZ1 is impaired; the tumor suppressor HIC1, MEN1 and CDC73 gene products, key molecules in parathyroid tumorigenesis, may be involved in epigenetic aberrant changes. Epigenetic changes are more frequent and more consistent in parathyroid malignancies, and positively correlated with severity of primary hyperparathyroidism.

HBx represses RIZ1 expression by DNA methyltransferase1 involvement in decreased miR-152 in hepatocellular carcinoma.

HepatitisB virus (HBV) is mainly suspected to promote hepatocellular carcinoma (HCC) development by epigenetic alteration. The HBV X protein (HBx) plays a key role in the molecular pathogenesis of HBV-related HCC. However, the mechanism of HBx-mediated hepatocarcinogenesis remains to be elucidated. RIZ1 gene, a candidate HCC suppressor gene, is frequently found to be hypermethylated and downregulated in HCC. In the present study, we show that the expression of RIZ1 was downregulated in 65% HCC tissues. Decreased expression of RIZ1 was restored by 5'-Aza in MHCC-97H HCC cell lines. HBx recombinant transfection increased DNMT1 expression level and suppressed RIZ1 expression. Moreover, knockdown of DNMT1 by siRNA restored RIZ1 expression in HCC cell SMMC-7721 and reduced methylated CpG sites of RIZ1. ChIP results showed that DNMT1 protein could bind to RIZ1 promoter, and this interaction was further enhanced with the transfected HBX recombinant. Moreover, miR-152 was decreased and involved in upregulation of DNMT1 in HBx transfected cells, at least partly, contributed to the epigenetic inactivation of RIZ1. Taken together, our data found that HBx repressed RIZ1 expression via DNMT1, which offered a new mechanism of RIZ1 inactivation in HCC, except for the widely known DNA methylation. These results enriched the epigenetic mechanism by which HBx contributes to pathogenesis of HBV-HCC.

Retinoblastoma protein interacting zinc finger 1 gene in hematological malignancies

Background: Several lines of evidence have shown that inactivation of tumor suppressor genes is closely associated with tumorigenesis in a wide variety of hematological malignancies. The role of downexpression of the RIZ1 gene in hematological malignancy has been reported in many studies, but its levels after receiving induction therapy in these patients have not been evaluated, which was the aim of this study. Objective: The current study aimed to find the relationship between expression of RIZ1 in cases of hematological malignancies before the start of induction therapy and at the end of induction therapy and its possible role as a risk factor in disease progression. Patients and methods: The study included two groups of patients: group I included 26 newly diagnosed patients with hematological malignancies before the start of induction therapy (day 0) and group II included 12 patients at the end of induction therapy (day 28). Group III included 10 individuals with another indication for bone marrow (BM) (such as peripheral blood unexplained cytopenia) who were age and sex matched with the patients and were enrolled in the study as a control group. The following investigations were performed for all the participants: complete blood count, BM sample for morphological examination, immunophenotyping, and cytogenetic analysis. The levels of RIZ1 gene expression were detected by real-time PCR (RT-qPCR) at diagnosis (day 0) and after induction therapy (day 28). Results: The mean levels of RIZ1 gene expression in day 0 patients were significantly lower than those of the control group, and at the end of induction therapy (on day 28), the level was significantly increased to reach close to that in the healthy controls. Furthermore, the levels of the RIZ1 gene were negatively correlated with the blast cell counts in peripheral blood and BM. Conclusion: We conclude that the RIZ1 gene is downexpressed in leukemic patients and its levels increased after induction therapy, indicating its possible role in disease pathogenesis. The relationship between its level and risk factors suggests its role in disease progression. A large-scale study is recommended for the use of the RIZ1 gene in the therapy of hematologic malignancies.

The unusual yin-yang fashion of RIZ1/RIZ2 contributes to the progression of esophageal squamous cell carcinoma

AbstractRetinoblastoma protein-interacting zinc finger gene RIZ encodes two different protein products, RIZ1 and RIZ2. Observations suggest that RIZ1 is a tumor suppressor, while RIZ2 acts as a negative regulator of RIZ1 and may play a positive role in oncogenesis. The imbalance amount of RIZ1 and RIZ2 may be involved in cancer development. In this study we detected the expression levels of RIZ1 and RIZ2 mRNA in human esophageal squamous cell carcinoma (ESCC) tissue specimens, reexpressed RIZ1 in the human ESCC cell line EC109 in which RIZ1 mRNA level was not detected, examined the changes of RIZ1 and RIZ2 mRNA expression, investigated the changes of proliferation, and apoptosis of the cells. We found that RIZ1 mRNA expression is commonly decreased or at undetectable level in human esophageal squamous cancer tissue specimens compared to the normal tissue specimens, while RIZ2 is usually expressed. With the forced expression of RIZ1, RIZ2 mRNA expression did not change, The ESCC cell proliferation was inhibited and apoptosis was induced. This unusual yinyang fashion of RIZ1/RIZ2 may contribute to the progression of ESCC.

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.

The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.

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.

Contribution of RIZ1 to Regulation of Proliferation and Migration of a Liver Fluke-Related Cholangiocarcinoma Cell

Retinoblastoma-interacting zinc finger gene (RIZ1) is a tumor suppressor gene which is highly inactivated by promoter hypermethylation in patients with liver fluke-related cholangiocarcinoma (CCA). Epigenetic aberration of this gene might withdraw the ability to restrain tumor cell proliferation and migration. We aimed to define the role of RIZ1 on cell proliferation and migration in CCA cell line. Small interference RNA (siRNA) was used to knock down the expression of RIZ1 in a CCA-derived cell line in which cell proliferation and cell migration were performed. A predominant nuclear localization of RIZ1 was observed. Reduction of RIZ1 by siRNA augmented cell proliferation and migration. The result suggested that RIZ1 might play a role in regulating cell proliferation and migration in CCA. Reduction of RIZ1 expression may aggravate the progression of CCA.

Epigenetic inactivation of the tumor suppressor gene RIZ1 in hepatocellular carcinoma involves both DNA methylation and histone modifications

The retinoblastoma-interacting zinc finger gene RIZ1 is inactivated in many cancers, but the underlying mechanisms remain unknown. This study aimed to investigate the epigenetic mechanisms of RIZ1 inactivation by analyzing the relationship between DNA methylation and histone modifications during regulation of RIZ1 expression. Methylation-specific PCR, RT-PCR, and immunohistochemistry were performed to examine RIZ1 methylation and expression. Dynamic changes in histone H3 lysine 9 (H3K9) modifications and histone deacetylases (HDACs) associated with the promoter were analyzed by chromatin immunoprecipitation (ChIP). RIZ1 methylation was detected in 66.7% (32/48) HCC tissues, 6.3% (3/48) corresponding non-cancerous tissues, and 66.7% (4/6) HCC cell lines. All 32 HCC tissues with promoter methylation showed complete loss of RIZ1 protein, whereas RIZ1 protein was present in all the corresponding non-cancerous tissues. Neither 5-aza-2-deoxycitidine (5-Aza-dC) nor Trichostatin A (TSA) reversed promoter methylation, but did restore RIZ1 mRNA and resulted in the downregulation of HDAC1 but not HDAC3. However, 5-Aza-dC+TSA induced a partial reversal of promoter methylation and a markedly synergistic reactivation of RIZ1. Moreover, both HDAC1 and HDAC3 were downregulated. The ChIP assays showed 5-Aza-dC and/or TSA also contributed to the dynamic conversion of trimethylated to acetylated H3K9 at the promoter. Furthermore, a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation. Our results suggest that promoter methylation and H3K9 modifications work together to silence the RIZ1 gene in HCC. 5-Aza-dC can restore the expression of RIZ1, as reflected by its effects on histone modification levels. This finding indicates that cooperative effects between these epigenetic modifications exist.

Effects of triptolide on RIZ1 expression, proliferation, and apoptosis in multiple myeloma U266 cells

To investigate the effects of triptolide on proliferation and apoptosis as well as on the expression of RIZ1 in the human multiple myeloma cell line U266 in vitro. The effect of triptolide on the growth of U266 cells was studied by MTT assay. Apoptosis was detected by Hoechst 33258 staining and Annexin V/PI double-labeled flow cytometry, and caspase-3 mRNA was measured by RT-PCR. Western blotting, flow cytometry and RT-PCR were used to assess the expression of RIZ1, and the location and expression of H3K9me1 were detected by confocal microscopy and Western blotting. Triptolide significantly inhibited the proliferation of U266 cells in a time- and concentration-dependent manner (the IC(50) value for a 24-h exposure was 157.19+/-0.38 nmol/L). Triptolide induced typical apoptotic morphological changes. Triptolide 40, 80, and 160 nmol/L treatment induced significant caspase-3-dependent apoptosis compared with control group (10.5%+/-1.23%, 37.9%+/-2.45%, and 40.5%+/-2.30% vs 3.8%+/-1.98%, P<0.05). Compared with peripheral blood monocular cells (PBMC) from healthy donors, the protein expression of RIZ1 in U266 cells was relatively low, but the mRNA and protein expression of RIZ1 were strikingly increased by triptolide in a concentration-dependent manner. Triptolide increased the protein expression of RIZ1 and RIZ1 methylates histone H3 lysine 9 in U266 cells. Triptolide increased the protein expression of RIZ1, inhibited the proliferation, and induced caspase-dependent apoptosis in U266 cells.

DNA methylation of the RIZ1 tumor suppressor gene plays an important role in the tumorigenesis of cervical cancer

AbtractObjectiveThe retinoblastoma protein-interacting zinc finger gene RIZ1 is a tumor suppressor gene and a member of a nuclear histone/protein methyltransferase superfamily. The purpose of the present study was to examine the expression of RIZ1 and evaluate its carcinogenesis in cervical cancer. The relationship between DNA methylation and transcriptional silencing of RIZ1 was investigated in cervical cancer.MethodsRIZ1 expression was examined in cervical cancer cell lines and cervical tissues (12 normal and 40 cancerous) by using RT polymerase chain reaction (PCR). Methylation status of the RIZ1 promoter was studied using methylation-specific PCR (MSP).ResultsRIZ1 expression is reduced or lost in cervical cancers, compared with normal cervical tissues (P < 0.05). The current study results also showed that loss of RIZ1 is mediated by aberrant cytosine methylation of the RIZ1 promoter. 37.5% of carcinomas were methylated, while none of normal tissues were methylated. RIZ1 mRNA expression was significantly higher (P = 0.000) in unmethylated (0.3494 ± 0.0466, mean ± SD), compared with methylated tissues (0.1422 ± 0.1073, mean ± SD). Treatment with a DNA methyltransferase inhibitor led to reactivation of RIZ1 expression in cell lines that had negligible RIZ1 expression at baseline.ConclusionsReduced expression of RIZ1 may play an important role in the pathogenesis and/or development of cervical cancer, and is considered to be caused in part by aberrant DNA methylation.

Correlation of Altered Expression of RIZ1 to Diagnosis, Risk Stratification, and Disease Progression in 56 Acute Leukemia Patients.

Abstract 2627Poster Board II-603RIZ1 expression is altered in a variety of human cancers and is now considered to be a candidate tumor suppressor in many types of human tumors, including breast cancer, liver cancer, colon cancer, neuroblastoma, melanoma, lung cancer, and osteosarcoma. Previous studies indicate that suppression of RIZ1 expression may have an important role in leukemogenesis and RIZ1 is downregulated during CML progression and displays tumor suppressor properties in CML cell lines. To deeply investigate the expression of RIZ1 and its correlation to diagnosis, risk stratification, and disease progression in human acute leukemia, we analysed the differential expression of RIZ1 by quantitative real-time reverse-transcription polymerase chain reaction assay in 56 acute patients with or without complete remission and in 9 healthy donors. Our results showed that the expression of RIZ1 was significantly decreased in both de novo acute leukemia patients and patients with no remission (n = 35) (P = 0.009, compared with healthy donors) and significantly increased in acute leukemia patients with complete remission (P = 0.001, compared with de novo acute leukemia patients and patients with no remission). When compared with healthy donors, acute leukemia patients with complete remission (n = 21) owned the statistically same expression of RIZ1 (P = 0.595). The expression of RIZ1 was significantly higher in acute myeloid leukemia (AML, n = 17) than acute lymphoblastic leukemia (ALL, n =15) (P = 0.009), with a good correlation to diagnosis of AML or ALL (r = 0.514). The patients (n = 16) with high-risk leukemia had significantly higher expression of RIZ1 than the patients with standard-risk (n = 18) (P = 0.031), with the correlation (r = 0.431) of RIZ1 expression to disease risk-stratification. In addition, the weak correlations of RIZ1 expression were shown to both age and gender ( r = 0.158 and 0.003, respectively). This relatively simple analysis provides an important role of RIZ1 expression in acute leukemia. Taken together, our results suggest the potential for using RIZ1 expression in predication of disease progression or recovery in acute leukemia. We demonstrate that RIZ1 expression can contribute to the diagnosis of AML or ALL and the evaluation of risk-stratification in acute leukemia. Disclosures:No relevant conflicts of interest to declare.

Decreased expression of RIZ1 and its clinicopathological significance in epithelial ovarian carcinoma: Correlation with epigenetic inactivation by aberrant DNA methylation

The retinoblastoma protein-interacting zinc finger gene (RIZ1) is considered a tumor suppressor gene. The purpose of the present study was to examine the expression of RIZ1 and evaluate its clinicopathological significance in ovarian carcinoma. Immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed for RIZ1 and its clinicopathological significance was examined. DNA methylation status of RIZ1 was also studied. All (6/6) of the normal, 5/9 of benign, and 4/9 of borderline tissues were positive for RIZ1 protein. In ovarian cancer tissues 32.9% (54/164) were positive for RIZ1. Decreased expression of RIZ1 was significantly correlated with histological subtypes (P < 0.0001), high tumor grade (P = 0.0153) and advanced clinical stage (P = 0.0345), and high Ki67 index (P = 0.0117) but was not associated with the overall prognoses of the patients (P = 0.519). The presence of methylated band was detected in 2/9 cell lines, and 5/69 ovarian cancer tissues. Median values of relative RIZ1 expression in cell lines with methylation were significantly lower than those without methylation (P = 0.0404), and treatment of 5-aza-2'deoxycitidine resulted in demethylation and re-expression of RIZ1. Reduced expression of RIZ1 may play an important role in the pathogenesis and/or development of epithelial ovarian carcinoma, and is considered to be caused in part by aberrant DNA methylation.

RIZ1 Is Downregulated during CML Progression and Displays Tumor Suppressor Properties in CML Cell Lines.

Transition to CML blast crisis is characterized by the continued presence of the Philadelphia chromosome and the acquisition of additional molecular and chromosomal alterations. Loss of heterozygosity at chromosome region 1p36 is a frequent finding in CML blast crisis. RIZ1 is located at 1p36 and frequently undergoes deletion, rearrangements, and loss of heterozygosity in a variety of cancers. Taken together, these data suggest that decreased RIZ1 expression may contribute to CML progression. We used immunohistochemistry to analyze RIZ1 expression in matched bone marrow biopsy specimens from CML chronic phase patients that progressed to accelerated phase or myeloid blast crisis. Similar to control bone marrow, strong cytoplasmic and nuclear RIZ1 expression was observed during chronic phase in all cases. However, RIZ1 expression was found to be markedly decreased in matched bone marrow biopsy specimens obtained during myeloid blast crisis in five patients. RIZ1 expression was maintained in the immature cells of two CML patients, one in accelerated phase with 15% myeloid blasts and the other in myeloid blast crisis, indicating that low RIZ1 expression is not an inherent property of immature hematopoietic cells. To confirm this, we analyzed G-CSF mobilized peripheral blood by flow cytometry and found RIZ1 to be expressed in mature myeloid and CD34+ cells. Transient transfection of myeloid blast crisis cell lines K562, YN-1, ERY-1, and JURL-MK1 with a RIZ1 expression plasmid (pRIZ1) increased the number of cells undergoing early and late apoptosis and reduced viability by 20–80% within 24 hours. RIZ1 effects on erythroid differentiation were assessed in K562, YN-1, and ERY-1 as these cell lines express low levels of hemoglobin, reflecting their myeloid/erythroid progenitor phenotype. As transient RIZ1 expression in these cells is too toxic to measure erythroid differentiation, we modified K562 to express less toxic levels of RIZ1 by stably integrating RIZ1 under the control of a CMV promoter (K562+RIZ1). As determined by benzidine staining, stable expression of RIZ1 in K562+RIZ1 increased erythroid differentiation compared to K562 alone. To confirm that RIZ1 is responsible for the enhanced erythroid differentiation, we transfected K562+RIZ1 as well as ERY-1 and YN-1 (which have higher endogenous RIZ1 levels than K562), with a plasmid that expresses RIZ1 shRNA (pRIZ1shRNA). Expression of pRIZ1shRNA in K562+RIZ1 reduced RIZ1 protein expression and erythroid differentiation to levels similar to that observed in K562 and decreased erythroid differentiation in ERY-1 and YN-1. RIZ1 effects on JURL-MK1 differentiation were assessed by measuring CD33 and CD117 as the expression of these proteins decreases during myeloid differentiation. Transient transfection of JURL-MK1 with pRIZ1 decreased CD33 and CD117 expression as assessed by flow cytometry. In summary, our study demonstrates that RIZ1 expression is frequently reduced in myeloid blast crisis. Furthermore, RIZ1 expression decreases cell proliferation, increases apoptosis, and enhances differentiation in cell line models of CML myeloid blast crisis. Taken together, our results build upon previous observations that a putative CML tumor suppressor gene is present at 1p36 and suggest that deregulation of RIZ1 expression may contribute to CML progression.

Identification of RIZ1 Targets Involvedin Erythroid Differentiation of K562 Human Erythroleukemia Cells Using Surface-Enhanced Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (SELDI).

RIZ1 (PRDM2) is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 functions as a tumor suppressor gene in a number of human cancers and is down regulated in some human acute myeloid leukemias. We previously found RIZ-1 to be silenced in K562 erythroleukemia cells by promoter hypermethylation. Furthermore, expression of RIZ1 in K562 promotes erythroid differentiation and also potentiates TGF-β1 mediated differentiation. To investigate similarities between genes altered by RIZ1 expression and the TGF-β1 pathway, we used SELDI to compare the protein profiles of K562 against K562 + RIZ1 and K562 + TGF-β1. Protein extracts for SELDI profiling were separated into six fractions according to their isoelectric points. Proteins from each fraction were then bound to two different protein chip surfaces (H50-hydrophoboic and CM10-cation exhange) and their mass/charge determined using SELDI. We analyzed four replicates from each sample and classified proteins as differentially expressed if their P-values were below 0.05. In total, we observed 104 differentially expressed proteins (60 upregulated and 44 down regulated) between K562 and K562 + RIZ1 and 176 proteins (96 upregulated and 80 down regulated) between K562 and K562 + TGF-β1. We used 2D-PAGE to identify differentially expressed proteins identified by SELDI analysis and located 48 proteins that were over expressed in K562 + RIZ1 and K562 + TGF-β1 relative to K562. To establish whether these proteins were the same proteins observed using SELDI, we determined if the proteins had the same pI and molecular weight and if the gel-eluted proteins bound to the same protein chip surface with the same mass/charge. 15 of 48 proteins passed the above criteria and we determined their identities using Trypsin-based peptide mapping strategies with molecular weight and pI restrictions. We identified two candidate proteins (14-3-3ε and S100/A13) that are similarly over expressed in K562 + RIZ1 and K562 + TGF-β1. These proteins have been shown to be associated with TGF-β1 signaling. Schistosomal 14-3-3ε interacts with SmRK1, a divergent type I transforming growth factor β1 receptor (TR-I) present on the surface of adult parasites and also binds to and activates human TR-I. S100/A13 belongs to a family of low molecular weight proteins characterized by the presence of two calcium-binding EF-hand motifs that includes S100C/A11, a member recently shown to play a key role in a PKCα mediated pathway essential for the growth inhibition of normal human keratinocytes by TGF-β1. In summary, we demonstrate the potential for using SELDI to identify novel proteins involved in regulating and connecting cellular growth and differentiation pathways.

Identification of Target Genes for the Tumor Suppressor RIZ1 Using Gene Expression Profiling.

RIZ1 (PRDM2) is a tumor suppressor gene on 1p36 that frequently undergoes deletion, rearrangements, and loss of heterozygosity in a broad spectrum of tumors. RIZ1 is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 contains a ~130 amino acid conserved domain (PR or SET) that is important in chromatin-mediated regulation of gene expression and in the development of cancer. RIZ1 methylates Histone H3 on K9 and this activity may play a role in transcription repression as H3-K9 methylation is known to be associated with repression. Aberrant activities or mistargeting of chromatin modifying activities are proving to have unexpected links to cancer. We and others have shown that RIZ1 expression is down regulated in human leukemias and in the human erythroleukemia cell line K562. Expression of RIZ1 in K562 reduced proliferation, increased apoptosis, and promoted erythroid differentiation. To understand how RIZ1's DNA binding, methyltransferase, and transcription repressor functions are related to its tumor suppressor activity it is necessary to characterize RIZ1 target genes. We used DNA microarrays to globally monitor how RIZ1 affects gene expression profiles. We constructed a K562 cell line with RIZ1 stably integrated under the control of a CMV promoter and analyzed the gene expression profiles of K652 and K562 + RIZ1 using a 42K Stanford human gene microarray. By comparing the gene expression profiles of these cell lines, we identified potential RIZ1 gene targets that are up and down regulated in the presence of RIZ1. In total, we identified 5 upregulated genes and 20 down regulated genes using significance analysis of microarrays (SAM) and standard deviation filter analysis of the gene expression data. RIZ1-mediated changes in gene expression profiling indicate that RIZ1 is potentially involved in the regulation and connection of the IGF-1 (IGF-1, IGFBP2) and integrin (LMS1) pathways, and in the activation of the TGF-β (SPARC) pathway. The genes perturbed by RIZ1 expression suggest that the tumor suppressor properties of RIZ1 arise from its control of proliferation, apoptosis and differentiation using these pathways. Finally, we observed an overrepresentation of the SP-1 transcription factor binding sites in genes that are upregulated in the absence of RIZ1. This correlates with the ability of RIZ1 to recognize SP1 sequences.

Altered expression of retinoblastoma protein-interacting zinc finger gene, RIZ, in human leukaemia.

The retinoblastoma protein-interacting zinc finger gene (RIZ), a member of the nuclear protein methyltransferase superfamily, is characterized by the presence of the N-terminal PR domain. The RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the PR (PRDI-BF1 and RIZ homologous) domain, RIZ2 lacks it. RIZ gene expression is altered in a variety of human cancers and RIZ1 is now considered to be a candidate tumour suppressor. To investigate the role of RIZ in leukaemogenesis, we analysed the differential expression of RIZ1 and RIZ2 by quantitative real-time reverse-transcription polymerase chain reaction assay. Our results showed that the expression of RIZ1 was significantly decreased in leukaemia cell lines (14 out of 17, 82%) and in patients with acute myeloblastic leukaemia (eight out of 14, 57%). In contrast, RIZ2 expression was increased in patients with acute lymphoblastic leukaemia (eight out of 11, 73%), compared with normal bone marrow cells. These findings indicate that suppression of RIZ1 expression or enhancement of RIZ2 expression may have an important role in leukaemogenesis.