Centrosome Aberrations in Bone Marrow Cells From Patients with Myelodysplastic Syndromes Correlate with Chromosomal Instability

Abstract Abstract 3839 Introduction: Centrosomes play important roles in maintenance of genetic stability and centrosomal aberrations are a common hallmark of cancer. Deregulation of centriole duplication during the cell cycle leads to supernumerary centrosomes, sister chromatide missegregation and could result in chromosomal instability (CIN) and aneuploidy. CIN is a common feature in at least 50% of patients with myelodysplastic syndromes (MDS). Therefore, we sought to investigate the centrosomal status and its role for development of CIN in bone marrow (BM) cells of MDS patients. Furthermore, deregulation of the protease Separase is known as a driver of aneuploidy. It is considered as one of the master key players in centriole duplication and overexpression has been associated with the formation of supernumerary centrosomes in many cancers. Therefore, deregulated Separase could also serve as a marker for genetic instability and was investigated. Patients and methods: BM cells of 34 MDS patients were cytogenetically examined by G-banding technique. Furthermore, cells were immunostained with a centrosome-specific antibody to pericentrin followed by a Cy3-conjugated secondary antibody to analyze the centrosomal status. Umbilical cord blood specimens (CB; n=15) served as controls. In addition, Separase protein levels were analyzed in BM cells of four MDS patients and in CB cells of four healthy controls. Results: BM cells of all MDS patients displayed centrosome alterations as compared with corresponding controls. Centrosome abnormalities were detected in 10% (range, 4–17%) of analyzed cells of MDS patients but in only 2% (range, 0–4%) of cells of healthy donors (p≤0.0001). Normal karyotypes were found in all CB metaphases and in BM metaphases of 16/34 MDS patients. The incidence of centrosomal alterations was higher in bone marrow cells of patients with cytogenetic alterations (mean, 12%) compared to BM cells of patients without cytogenetic changes (mean, 7%). In BM cells of MDS patients Separase protein levels were lower (60% decrease) as compared to CB cells of the healthy control (p≤0.01). Conclusions: We could show that centrosome aberrations in BM cells of MDS patients occur before chromosomal changes are detectable. Therefore, centrosomal instability is an early step in MDS and may contribute to the acquisition of chromosomal alterations. Increase of aberrant centrosomes significantly correlates with karyotype instability and aneuploidy. It seems that centrosomal instability precedes karyotype instability via chromosomal missegregation and could contribute to the development of chromosomal changes and accelerate malignant transformation. Separase is one of the master key players in centriole duplication and chromatide segregation. Therefore, deregulated Separase could lead to genetic instability and malignant transformation. In future studies centrosomal alterations may serve as an additional prognostic biomarker for future diagnostics in MDS. Disclosures: No relevant conflicts of interest to declare.

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Abnormal PI 3-Kinase Activity Due to Increased Lyn with Decreased PTEN and Absent SHIP in Bone Marrow Cells from Patients with Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v106.11.3446.3446 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3446-3446

Author(s):

Daniel W. Lee ◽

Quan-Sheng Zhu ◽

Sonali Rudra ◽

Elizabeth J. Shpall ◽

Steven Kornblau ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Kinase Activity ◽

Bone Marrow Cells ◽

Constitutive Activation ◽

Protein Levels ◽

Inositol Ring ◽

Marrow Cells

Abstract Myelodysplastic syndromes (MDS) result from a malignant stem cell clone characterized by ineffective hematopoiesis, manifested as peripheral cytopenia with a cellular bone marrow. A leading hypothesis is that MDS results from a breakdown in the control of myeloid cell proliferation and apoptosis. Through its generation of 3′-phosphoinositides and subsequent activation of effectors such as Akt, phosphatidylinositol 3-kinase (PI 3-kinase) drives cell proliferation, apoptosis, differentiation, and motility. We show here that PI 3-Kinase is profoundly deregulated in high-risk MDS. Bone marrow cells from high-risk MDS patients displayed a 3–30 fold increase in constitutive activation of the Src kinase Lyn. Constitutive serine/threonine phosphorylation of Akt, a surrogate of PI-3 kinase activity, was seen in all specimens. Protein levels of PTEN, which dephosphorylates the D3-position phosphate of the inositol ring, were variably decreased. Since PTEN is frequently silenced by hypermethylation, we treated U937, THP-1, and Mo7e cells with 5-azacytidine. However, real-time PCR showed no increase in PTEN transcripts in these cell lines. More significantly, protein expression of SHIP-1, which dephosphorylates the D5-position phosphate of the inositol ring, was markedly decreased or absent in bone marrow cells from MDS patients, whereas they were present in AML or ALL blasts. Real-time PCR showed SHIP-1 transcripts in MDS cells to be 50% of normal CD34+ stem cells. Although 5-azacytidine treatment resulted in an increase in SHIP-1 transcripts, as measured by quantitative PCR, protein levels did not increase. Because SHIP-1 contains three PEST-rich regions, we hypothesized that the low or absent level of expression of SHIP-1 may due to protein degradation. Neither the cryopreservation of cells nor lysis buffer could explain the absence of SHIP-1 protein. Instead, when U937 and HL60 leukemic cell lines were treated with the proteastome inhibitor lactacystin (1–5 uM), protein levels of SHIP-1 increased. These results suggest that constitutive activation of Akt is likely due to decreased PTEN and absent SHIP-1 protein levels in primary MDS cells. Consistent with these findings, mice deficient in PTEN and SHIP-1 suffer from anemia, thrombocytopenia, leukocytosis and impaired function of myeloid progenitors (BLOOD103:4503, 2004). These results also suggest that a combination of a Src kinase inhibitor and a proteasome inhibitor may be a therapeutic approach for the treatment of high-risk MDS.

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