CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins

Abstract

CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) are distinguished from other MDS cells and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We previously demonstrated up-regulation of c-myc and CD1 by microarray analysis. Here, we confirmed these findings by real-time polymerase chain reaction (PCR), demonstrated up-regulation of survivin, c-myc, and CD1 protein expression, and documented comparable colony formation by annexin(+) trisomy 8(-) CD34(+) and annexin(-) CD34 cells. There were low levels of DNA degradation in annexin(+) trisomy 8 CD34 cells, which were comparable with annexin(-) CD34 cells. Trisomy 8 cells were resistant to apoptosis induced by gamma irradiation. Knock-down of survivin by siRNA resulted in preferential loss of trisomy 8 cells. These results suggest that trisomy 8 cells undergo incomplete apoptosis and are nonetheless capable of colony formation and growth.

Figure 1

Up-regulation of CD1 and c-myc in CD34⁺ cells of patients with trisomy 8. CD34⁺ cells from the BM of patients with trisomy 8 were separated by flow cytometry, and mRNA was extracted and subjected to real-time PCR as described in “Materials and methods”; FISH was also performed to determine the percentage of trisomy 8 cells in the same sample. Both c-myc and CD1 m-RNAs were up-regulated in trisomy 8 compared with a control group of 10 healthy donors (A). The magnitude of increase in c-myc and CD1 expression was proportional to the number of trisomy 8 CD34 cells in the sample (B-C). Error bars represent standard error of the mean (SEM).

Figure 2

CD1 protein is overexpressed in trisomy 8. BMMNCs were stained with CD1-FITC and CD34-PE mAb and subjected to flow cytometry; CD34⁺ cells were gated, and the number of these cells staining with anti-CD1–FITC was quantitated. CD1 protein was increased in CD34 cells from patients with trisomy 8 compared with a group of healthy controls (A). CD1 expression was proportional to the number of trisomy 8 CD34 cells in the sample as determined by FISH using a specific chromosome 8 probe. (B) BMMNCs from patients with trisomy 8 were stained with CD34-PE and CD1-FITC. Cells were sorted into CD34⁺, CD1⁺, and CD1⁻ aliquots by flow cytometry; FISH for chromosome 8 was performed on the sorted samples. There were increased trisomy 8 cells in the CD1⁺ fraction. (C) Immunoblots were performed on nuclear extracts of BMMNCs for c-myc. Increases in patients with trisomy 8 compared with healthy controls (n = 2) or other patients with MDS (n = 6) were seen. Error bars represent SEM.

Figure 3

Survivin, an IAP, is up-regulated in trisomy 8. RNA was extracted from purified CD34 cells with trisomy 8 and analyzed for survivin by real-time PCR; FISH was performed on sorted samples. Expression was compared with the mean of a panel of 10 healthy donors, as described in “Materials and methods.” Survivin mRNA was up-regulated in patients with trisomy 8 compared with controls and with a small group of equally cytopenic patients whose cytogenetics showed monosomy 7 (A). Expression was increased to a greater extent in samples with increased trisomy 8 cells (B). Error bars represent SEM.

Figure 4

Survivin protein is increased in trisomy 8 CD34 cells. Flow cytometry was performed on column-purified CD34 cells stained by intracellular staining as described in “Materials and methods” using survivin-FITC mAb. Survivin protein was increased in samples obtained from trisomy 8 BM (example seen in panel A). Entire sampling of patients examined is seen in panel B. Immunoblots were performed on total-cell lysates of BMMNCs for survivin. Increases in patients with trisomy 8 compared with healthy controls (n = 2) or other patients with MDS (n = 6) could be seen. Error bars represent SEM.

Figure 5

Absence of significant DNA degradation in annexin⁺ cells from patients with trisomy 8. (A) Cell-cycle analysis of sorted CD34 annexin⁺ and annexin⁻ cells showed that trisomy 8 cells were more heavily represented in the annexin⁺ fraction, but no significant numbers of cells with subgenomic DNA could be seen in this fraction (full dataset in Table 1). (B) When purified CD34 cells were stained with annexin-PE and CD34-FITC, or the TUNEL assay performed, there was significant annexin staining, but few cells were TUNEL⁺ (full dataset in Table 1).

Figure 6

Comparable hematopoietic colony formation by annexin⁺ and annexin⁻ CD34 cells from patients with trisomy 8. BMMNCs obtained from patients with significant trisomy 8 populations were stained with annexin-FITC and CD34-PE mAb and sorted into annexin⁺ and annexin⁻ aliquots of CD34 cells. These CD34 cells were plated in methycellulose for short-term culture as described in “Materials and methods.” Annexin⁺ CD34 cells were primarily trisomy 8 and gave rise to more hematopoietic colonies than did annexin⁻ cells in 3 of 4 patients. In all 5 samples obtained from healthy volunteers, the annexin⁺ CD34 cells failed to give rise to any colonies. FISH results on annexin⁺ and annexin⁻ fractions are listed in Table 1. Error bars represent SEM.

Figure 7

Resistance of trisomy 8 cells to apoptosis is abrogated by knock-down of c-myc and survivin.Trisomy 8 BM was subjected to 25 Gy of gamma radiation and cultured for 1 hour in media as described in “Materials and methods.” The TUNEL assay was performed to identify DNA nicks and TUNEL⁺ and TUNEL⁻ fractions sorted for both irradiated and nonirradiated fractions. The numbers of viable cells were decreased in the irradiated fraction, and the TUNEL⁺ cells increased from 18% to 30%. When FISH was performed on these fractions, trisomy 8 cells were consistently present in the TUNEL⁺ fraction (A). When control (nonirradiated) and irradiated lymphocyte-depleted BMMNCs were placed in short-term methylcellulose culture for 2 weeks the numbers of diploid colonies decreased to a greater extent than the numbers of trisomy 8 colonies (B). When an inhibitor of c-myc was cocultured with BMMNCs from a patient with trisomy 8, and immunoblotted, and FISH performed after 2 weeks of growth, there was a substantial decrease in the number of trisomy 8 cells coincident with a decrease in c-myc protein (C). BMMNCs transfected with siRNA specific for survivin were placed in hematopoietic growth factors for 36 hours at 37°C as described in “Materials and methods.” FISH and cell enumeration were performed before, and following culture using a labeled probe for chromosome 8. Immunoblotting was performed on total-cell lysates using antibody to survivin. (D) A substantial loss of trisomy 8 cells is seen compared with the baseline.