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RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression.

Lakshmikuttyamma A, Takahashi N, Pastural E, Torlakovic E, Amin HM, Garcia-Manero G, Voralia M, Czader M, DeCoteau JF, Geyer CR - J Hematol Oncol (2009)

Bottom Line: Expression of RIZ1 CML blast crisis cell lines decreased proliferation, increased apoptosis, and enhanced differentiation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Stem Cell Research Group, University of Saskatchewan, Saskatoon, SK, Canada. ashakum@htmail.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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RIZ1 expression in bone marrow of CML patients. (a) Immunohistochemical analysis of matched bone marrow trephine biopsies and bone marrow aspirate clot samples from patients in chronic phase and accelerated phase or myeloid blast crisis using an anti-RIZ1 antibody. (b) RIZ1 expression in normal bone marrow and normal bone marrow staining in the absence of RIZ1 primary antibody (Negative control). (c) Immunohistochemical analysis of RIZ1 expression in unmatched patient bone marrow biopsies and clot sections from chronic phase (CP) (N = 10), accelerated phase (AP) (N = 7) and blast crisis (BC) (N = 15) using an anti-RIZ1 monoclonal antibody. Relative RIZ1 expression represents 3,3-diaminobenzidine chromagen intensity. Mean RIZ1 expression for each group is shown as a black line and errors bars represent the standard deviation.
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Figure 1: RIZ1 expression in bone marrow of CML patients. (a) Immunohistochemical analysis of matched bone marrow trephine biopsies and bone marrow aspirate clot samples from patients in chronic phase and accelerated phase or myeloid blast crisis using an anti-RIZ1 antibody. (b) RIZ1 expression in normal bone marrow and normal bone marrow staining in the absence of RIZ1 primary antibody (Negative control). (c) Immunohistochemical analysis of RIZ1 expression in unmatched patient bone marrow biopsies and clot sections from chronic phase (CP) (N = 10), accelerated phase (AP) (N = 7) and blast crisis (BC) (N = 15) using an anti-RIZ1 monoclonal antibody. Relative RIZ1 expression represents 3,3-diaminobenzidine chromagen intensity. Mean RIZ1 expression for each group is shown as a black line and errors bars represent the standard deviation.

Mentions: We characterized RIZ1 expression in matched bone marrow biopsies from seven CML patients in chronic phase, accelerated phase, or myeloid blast crisis by immunohistochemistry (Fig 1a). Anti-RIZ1 antibody is specific for the N-terminus of RIZ1 and thus does not recognize the RIZ2 isoform [6]. Previously this antibody has been used to specifically detect RIZ1 in flow cytometry [6], Western analysis [6], and chromatin immunoprecipitation assays [2,6]. We observed strong cytoplasmic and nuclear RIZ1 expression during chronic phase in all cases, which was similar to RIZ1 expression in normal bone marrow (Fig 1a, b). Five of six cases in blast crisis had markedly reduced RIZ1 expression (Cases 1–5). In Case 1, the patient had focal blast crisis and RIZ1 expression was stronger in areas not involved in the blast foci. One blast crisis patient (Case 7) and an accelerated phase patient (Case 6) showed no appreciable change in RIZ1 expression. To validate these results further, we analyzed RIZ1 expression in a CML tissue microarray containing a larger cohort of unmatched bone marrow biopsies in chronic phase, accelerated phase, and blast crisis by immunohistochemistry. We observed a significant decrease in RIZ1 expression (P = 0.015) in blast crisis compared to chronic phase biopsies (Fig 1c). We did not observe any significant differences in RIZ1 expression between chronic phase and accelerated phase or between accelerated phase and blast crisis. The mean value for RIZ1 expression in blast crisis separates high and low RIZ1 expressing biopsies and was approximately equal to the lower standard deviation for RIZ1 expression in chronic phase. This is consistent with other molecular defects in the high RIZ1 expression biopsies contributing to the chronic phase to blast crisis transition. Abnormalities of proto-oncogenes, such as RAS and MYC, or of tumor suppressor genes, such as mutations of the p53 gene, absence of RB protein, and homozygous deletions of the p16INK4a gene, have been reported to occur during the chronic phase to blast crisis transition [7].


RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression.

Lakshmikuttyamma A, Takahashi N, Pastural E, Torlakovic E, Amin HM, Garcia-Manero G, Voralia M, Czader M, DeCoteau JF, Geyer CR - J Hematol Oncol (2009)

RIZ1 expression in bone marrow of CML patients. (a) Immunohistochemical analysis of matched bone marrow trephine biopsies and bone marrow aspirate clot samples from patients in chronic phase and accelerated phase or myeloid blast crisis using an anti-RIZ1 antibody. (b) RIZ1 expression in normal bone marrow and normal bone marrow staining in the absence of RIZ1 primary antibody (Negative control). (c) Immunohistochemical analysis of RIZ1 expression in unmatched patient bone marrow biopsies and clot sections from chronic phase (CP) (N = 10), accelerated phase (AP) (N = 7) and blast crisis (BC) (N = 15) using an anti-RIZ1 monoclonal antibody. Relative RIZ1 expression represents 3,3-diaminobenzidine chromagen intensity. Mean RIZ1 expression for each group is shown as a black line and errors bars represent the standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: RIZ1 expression in bone marrow of CML patients. (a) Immunohistochemical analysis of matched bone marrow trephine biopsies and bone marrow aspirate clot samples from patients in chronic phase and accelerated phase or myeloid blast crisis using an anti-RIZ1 antibody. (b) RIZ1 expression in normal bone marrow and normal bone marrow staining in the absence of RIZ1 primary antibody (Negative control). (c) Immunohistochemical analysis of RIZ1 expression in unmatched patient bone marrow biopsies and clot sections from chronic phase (CP) (N = 10), accelerated phase (AP) (N = 7) and blast crisis (BC) (N = 15) using an anti-RIZ1 monoclonal antibody. Relative RIZ1 expression represents 3,3-diaminobenzidine chromagen intensity. Mean RIZ1 expression for each group is shown as a black line and errors bars represent the standard deviation.
Mentions: We characterized RIZ1 expression in matched bone marrow biopsies from seven CML patients in chronic phase, accelerated phase, or myeloid blast crisis by immunohistochemistry (Fig 1a). Anti-RIZ1 antibody is specific for the N-terminus of RIZ1 and thus does not recognize the RIZ2 isoform [6]. Previously this antibody has been used to specifically detect RIZ1 in flow cytometry [6], Western analysis [6], and chromatin immunoprecipitation assays [2,6]. We observed strong cytoplasmic and nuclear RIZ1 expression during chronic phase in all cases, which was similar to RIZ1 expression in normal bone marrow (Fig 1a, b). Five of six cases in blast crisis had markedly reduced RIZ1 expression (Cases 1–5). In Case 1, the patient had focal blast crisis and RIZ1 expression was stronger in areas not involved in the blast foci. One blast crisis patient (Case 7) and an accelerated phase patient (Case 6) showed no appreciable change in RIZ1 expression. To validate these results further, we analyzed RIZ1 expression in a CML tissue microarray containing a larger cohort of unmatched bone marrow biopsies in chronic phase, accelerated phase, and blast crisis by immunohistochemistry. We observed a significant decrease in RIZ1 expression (P = 0.015) in blast crisis compared to chronic phase biopsies (Fig 1c). We did not observe any significant differences in RIZ1 expression between chronic phase and accelerated phase or between accelerated phase and blast crisis. The mean value for RIZ1 expression in blast crisis separates high and low RIZ1 expressing biopsies and was approximately equal to the lower standard deviation for RIZ1 expression in chronic phase. This is consistent with other molecular defects in the high RIZ1 expression biopsies contributing to the chronic phase to blast crisis transition. Abnormalities of proto-oncogenes, such as RAS and MYC, or of tumor suppressor genes, such as mutations of the p53 gene, absence of RB protein, and homozygous deletions of the p16INK4a gene, have been reported to occur during the chronic phase to blast crisis transition [7].

Bottom Line: Expression of RIZ1 CML blast crisis cell lines decreased proliferation, increased apoptosis, and enhanced differentiation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Stem Cell Research Group, University of Saskatchewan, Saskatoon, SK, Canada. ashakum@htmail.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus