- Short report
- Open Access
RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression
© Lakshmikuttyamma et al; licensee BioMed Central Ltd. 2009
- Received: 17 March 2009
- Accepted: 14 July 2009
- Published: 14 July 2009
RIZ1 expression and activity are reduced in many cancers. In AML cell lines and patient material, RIZ1 expression is reduced relative to normal bone marrow. In chronic myelogenous leukemia (CML), blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located. RIZ1 is a PR domain methyltransferase that methylates histone H3 lysine 9, a modification important for transcriptional repression. In CML blast crisis cell lines RIZ1 represses insulin-like growth factor-1 expression and autocrine signaling. Together these observations suggest that RIZ1 may have a role in the chronic phase to blast crisis transition in CML.
In CML patient material, we observed that RIZ1 expression was decreased during progression from chronic phase to blast crisis. RIZ1 was expressed in mature myeloid and CD34+ cells demonstrating that decreased RIZ1 expression in blast crisis is not due to an increased immature cell population. Expression of RIZ1 CML blast crisis cell lines decreased proliferation, increased apoptosis, and enhanced differentiation.
RIZ1 is a candidate tumor suppressor gene whose expression is decreased in blast crisis. Loss of RIZ1 activity results in decreased apoptosis and differentiation and enhanced proliferation. Together these results suggest that loss of RIZ1 expression will lead to an increase in myeloid blast cell population resulting in CML progression.
- Chronic Phase
- Chronic Myelogenous Leukemia
- Blast Crisis
- Accelerate Phase
- Erythroid Differentiation
Molecular mechanisms responsible for driving the transition of chronic myelogenous leukemia (CML) from chronic phase to blast crisis are not well characterized. CML evolves from a chronic phase that is associated with the Philadelphia chromosome to a blast crisis phase, which is associated with additional chromosome or molecular aberrations. Evolution to blast crisis is correlated with frequent loss of heterozygosity at chromosome region 1p36 . RIZ1, a PR domain methyltransferase, is located at 1p36. RIZ1 methylates histone H3 lysine 9, a modification important for transcriptional repression . RIZ1 expression and activity are reduced in many human cancers by genetic and epigenetic mechanisms [3, 4]. RIZ1 expression is reduced in acute myeloid leukemia  and the RIZ1 knockout mouse has a high incidence of diffuse large B-cell lymphoma . RIZ1 also regulates IGF-1 signaling in CML blast crisis cell lines . Together these data suggest that decreased RIZ1 expression may contribute to CML progression. We investigated whether RIZ1 expression was reduced during CML progression and whether RIZ1 induced phenotypes that support its role as a candidate tumor suppressor.
The mechanism for decreased RIZ1 expression in CML blast crisis is not known. One possible explanation is that the RIZ1 promoter CpG island is aberrantly hypermethylated. In the CML blast crisis cell line, K562, the RIZ1 promoter is hypermethylated and addition of a methylation inhibitor, 5-aza-2'-deoxycytidine, induces RIZ1 expression . Epigenetic silencing has also been reported to reduce RIZ1 expression in other cancers .
We analyzed the effect of RIZ1 expression on megakaryocytic differentiation in JURL-MK1 cells by measuring changes in CD33 and CD117 using flow cytometry and immunocytochemistry. CD33 and CD117 are present in myeloid progenitors and their expression decreases with maturation and differentiation. Transient transfection of pRIZ1 into JURL-MK1 decreased CD33 and CD117 expression as monitored by flow cytometry (Fig 4e). Immunohistochemical staining using CD117 antibody also shows that transient transfection of pRIZ1 into JURL-MK1 decreased CD117 expression (Fig 4e).
These results build upon previous observations that a putative CML tumor suppressor gene is present at 1p36 that exhibits loss of heterozygosity during transformation from chronic phase to blast crisis . We propose a model whereby in chronic phase CML there is an expansion of BCR/ABL positive CML progenitor cells that maintain the ability to undergo apoptosis and differentiation. Epigenetic or genetic aberrations in RIZ1 expression and activity result in a blockage of apoptotic and differentiation pathways, which causes expansion of the myeloid blast cell population.
Cell Lines, CD34+ Cells, and CML Patient Material
K562 is from ATCC (Manassas, VA, USA), JURL-MK1 is from DSMZ (Braunschweig, Germany), YN-1, ERY-1, and K562+RIZ1 have been described previously . CD34+ cells were purified from G-CSF mobilized peripheral blood using an AutoMACs Separator with a Direct CD34 Progenitor Cell Isolation Kit from Miltenyi Biotech (Auburn, CA, USA). Fixed bone marrow specimens from CML chronic phase patients that progressed to accelerated phase or blast crisis were obtained from the Department of Pathology and Laboratory Medicine (Indiana University). Patients were diagnosed in chronic phase between 1997–2000 and in accelerated phase or blast crisis between 2000–2004. Unmatched patient CML bone marrow biopsies and clot sections were obtained from the MD Anderson Cancer Center as described previously . Patient samples were obtained with informed consent according to institutional review board guidelines.
Cell Line Transfections and Assays
Plasmids were transfected into cell lines using the Nucleofector system (Amaxa, Gaithersburg, MD, USA). Transfection efficiencies for CML cell lines were: K562 – 74.5% ERY-1 – 68.6%, YN-1 – 75.3, JURL-MK1 – 77%. pRIZ1 (p3RIZRH4.1) was from Steele-Perkins et al,  and pCDNA3 was from Invitrogen (Carlsbad, CA, USA). pRIZ1shRNA and shRNA non-silencing control vector were from OPEN Biosystems (Huntsville, AL, USA). Cell viability, apoptosis, and hemoglobin staining were assayed using Trypan blue dye exclusion, Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA), and benzidine staining, respectively.
Conjugated antibodies used for surface analysis of CD45, CD34, CD33, and CD117 expression are from Beckman Coulter (Fullerton, CA, USA). Intracellular RIZ1 expression was detected indirectly using anti-RIZ1 monoclonal antibody (1:25 dilution; Abgent, San Diego, CA, USA) and a FITC-conjugated secondary antibody following fixation and permeabilization with IntraPrep reagent (Beckman Coulter).
Immunohistochemical analysis of B5 fixed/paraffin embedded and decalcified bone marrow trephine biopsies and B5 fixed/paraffin embedded bone marrow aspirate clot samples was performed using an anti-RIZ1 monoclonal antibody (Abgent, San Diego, CA, USA) (1:25 dilution) and a horseradish peroxidase-coupled secondary antibody. RIZ1 expression in unmatched patient bone marrow biopsies and clot sections was calculated by measuring intensity levels of 3,3-diaminobenzidine chromogen staining (brown pixel intensity) that was normalized to the area scanned using an ACIS® III scanner (Dako, Carpinteria, CA, USA). Statistical differences between chronic phase, accelerated phase, and blast crisis were determined using an unpaired t-test.
Total RNA was isolated from cell lines using the TRI-zol reagent (Life Technologies). cDNA was synthesized from total RNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). cDNA was amplified in a 50 μl reaction containing Hotstar Taq DNA polymerase and buffer (Qiagen), 100 pmol primers (RIZ1: 5'-AACATGTGCTGCGAGGACTT-3' and 5'-TTCTTCCCTTTCCGGCTCT T-3'; β-Actin: 5' CCAAGGCCAACCGCGAGAAGAT-3' and 5'-TTGCTCGAAGTC CAGGGCGA-3'), and 0.25 μg cDNA.
All the data are reported as mean± s.d. The differences between the mean values were tested for statistical significance by the two-tailed Student's t-test (P-values).
A.L. is a Rethink Breast Cancer Research Fellow. E.P. is a CIHR Postdoctoral Research Fellow. C.R.G is a CIHR-RPP New Investigator. This work was supported by grants from the Canadian Cancer Society, Canadian Institutes of Health Research, Canadian Foundation for Innovation, and the Saskatchewan Health Research Foundation
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