Innovative analyses support a role for DNA damage and an aberrant cell cycle in myelodysplastic syndrome pathogenesis.

We used flow cytometry to analyze the cell cycle, DNA damage, and apoptosis in hematopoietic subsets in MDS marrow. Subsets were assigned using CD45, side scatter, CD34, and CD71. Cell cycle fractions were analyzed using DRAQ 5 (DNA content) and MPM-2 (mitoses). DNA damage was assessed using p-H2A.X, and apoptosis using Annexin V. Compared to controls, MDS patients demonstrated no increased mitoses in erythroid, myeloid, or CD34+ cells. Myeloid progenitors demonstrated increased G2 cells, which with no increased mitoses suggested delayed passage through G2. Myeloid progenitors demonstrated increased p-H2A.X, consistent with DNA damage causing this delay. Annexin V reactivity was equivalent in MDS and controls. Results for each parameter varied among hematopoietic compartments, demonstrating the need to analyze compartments separately. Our results suggest that peripheral cytopenias in MDS are due to delayed cell cycle passage of marrow progenitors and that this delayed passage and leukemic progression derive from excessive DNA damage.

Patient-derived acute myeloid leukemia (AML) bone marrow cells display distinct intracellular kinase phosphorylation patterns.

Multiparametric analyses of phospho-protein activation in patients with acute myeloid leukemia (AML) offers a quantitative measure to monitor the activity of novel intracellular kinase (IK) inhibitors. As recent clinical investigation with FMS-like tyrosine-3 inhibitors demonstrated, targeting IK with selective inhibitors can have a modest clinical benefit. Because multiple IKs are active in patients with AML, multikinase inhibitors may provide the necessary inhibition profile to achieve a more sustained clinical benefit. We here describe a method of assessing the activation of several IKs by flow cytometry. In 40 different samples of patients with AML we observed hyper-activated phospho-proteins at baseline, which is modestly increased by adding stem cell factor to AML cells. Finally, AML cells had a significantly different phospho-protein profile compared with cells of the lymphocyte gate. In conclusion, our method offers a way to determine the activation status of multiple kinases in AML and hence is a reliable assay to evaluate the pharmacodynamic activity of novel multikinase inhibitors.

A standardized ZAP-70 assay--lessons learned in the trenches.

BACKGROUND: The disease of chronic lymphocytic leukemia (CLL) has been shown to exhibit varying clinical outcomes based on reported laboratory parameters. One of these parameters involves the measurement of the protein levels of zeta-associated protein (ZAP-70) in CLL cells. A standardized assay has not yet reached consensus in the clinical cytometry community. METHODS: We developed a system using the 8-peak Rainbow beads as our fluorescence calibrator along with a fixed cell control. Using a panel of CD19-PE, CD5-FITC, and ZAP-70-Alexa 647, we stained normal whole blood, and blood and bone marrow from patients with CLL to determine the level of ZAP-70 expression in T-cell, B-cell, and CLL-cell populations. ZAP-70 expression was reported in molecules equivalent fluorescence (MEFL) based on the calibration of the flow cytometer with the 8-peak Rainbow beads. RESULTS: Daily assay performance as well as operating MEFL defined ranges for ZAP-70 detection in CLL were developed. A rank-order, nonparametric approach to reference ranges was used to assign a cutoff for "negative" as well as ranges for "intermediate" and "positive" staining using T and B cells from a pool of 50 normal subjects, and CLL cells from 395 patients. The assay was validated in a multi-institutional study and has demonstrated correlation with published techniques. Since its initial development, the assay has been implemented at two additional laboratory sites and has been shown to produce reproducible, correlated data at all sites. CONCLUSIONS: Strict adherence to standardization can yield an assay that is predictable, reliable, and reproducible as well as capable of multisite implementation. The Rainbow beads provide a common platform for system calibration. The fixed cell culture controls provide a common target to test antibody. The final level of control tests the sensitivity of ZAP-70 detection in a normal peripheral blood sample stained along with the submitted CLL patients. The acceptance/rejection of test results must meet all three levels of control before patient results are reported.

Dysregulated human myeloid nuclear differentiation antigen expression in myelodysplastic syndromes: evidence for a role in apoptosis.

Reduced levels of human myeloid nuclear differentiation antigen (MNDA) gene transcripts have been detected in both familial and sporadic cases of myelodysplastic syndromes (MDS). Numerous reports implicate elevated apoptosis/programmed cell death and death ligands and their receptors in the pathogenesis of MDS. MNDA and related proteins contain the pyrin domain that functions in signaling associated with programmed cell death and inflammation. We tested the hypothesis that MNDA is involved in the regulation of programmed cell death in human myeloid hematopoietic cells. Clones of K562 cells (MNDA-null) that expressed ectopic MNDA protein were established using retroviral transduction. MNDA-expressing K562 clones were resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, but were not protected from programmed cell death induced with genotoxic agents or H(2)O(2). MNDA protein expression assessed in control and intermediate and high-grade MDS marrows showed several patterns of aberrant reduced MNDA. These variable patterns of dysregulated MNDA expression may relate to the variable pathophysiology of MDS. We propose that MNDA has a role regulating programmed cell death in myeloid progenitor cells, and that its down-regulation in MDS is related to granulocyte-macrophage progenitor cell sensitivity to TRAIL-induced programmed cell death.

S100P is selectively upregulated in tumor cell lines challenged with DNA cross-linking agents.

Bifunctional alkylating agents that cross-link DNA are implicated in the pathogenesis of therapy related myelodysplastic syndromes (MDS) and MDS related acute myeloid leukemia (MDR-AML). We exposed HL60 cells to the highest level of bifunctional alkylating nitrogen mustard mechlorethamine (HN2) that was consistent with recovery following suppressed growth. Microarray analyses showed minor changes in transcripts in HN2 treated cells. A moderate up-regulation of S100P mRNA was consistently observed after 1 day of exposure to bifunctional alkylating agents and expression was not induced with monofunctional agents. Elevated S100P protein/antigen was not detected until days later in a subset of non-mitotic G2 cells. Elevated S100P protein persisted over the course of a delayed recovery phase. The results confirm recent reports indicating that S100P is a survival factor. In addition, our results indicate that S100P has a specific role in G2 cell function associated with a prolonged phase of recovery after exposure to bifunctional alkylating agents.

Overexpression of GSTA2 protects against cell cycle arrest and apoptosis induced by the DNA inter-strand crosslinking nitrogen mustard, mechlorethamine.

The effectiveness of bifunctional alkylating nitrogen mustard compounds in chemotherapy is related to their ability to form DNA inter-strand crosslinks. Patients exposed to DNA inter-strand crosslinking (ICL) agents subsequently experience an elevated incidence of myelodysplastic syndromes (MDS) and MDS related acute myeloid leukemia. Fanconi's anemia (FA) patients are deficient in the repair of crosslink DNA damage and they experience a high incidence of MDS. These observations indicate that hematopoietic cells are specific target for the transforming effects of DNA crosslinking damage. Changes in transcript levels were characterized in human hematopoietic cells occurring in response to the nitrogen mustard, mechlorethamine (HN2), but not in response to monofunctional analogs. Only modest changes in a few gene transcripts were detected in HL60 cells exposed to levels of HN2 tittered to maximal dose that caused growth suppression with minimal cell death and allowed eventual resumption of normal cell growth. Under conditions of transient growth suppression, a subset of glutathione-S-transferase (GST) isoenzyme genes was consistently upregulated three to fourfold by HN2, but not by monofunctional analogs. Subsequent efforts to confirm the changes detected by microarray analyses revealed an unexpected dependence on treatment conditions. The GST alpha class A2 subfamily member transcripts were upregulated 24 h after a 1 h exposure to HN2 that caused an extensive, but transient block in late S/G2 cell cycle phase, but were minimally altered with continuous exposure. The 1-h exposure to HN2 caused a transient late S/G2 cell cycle arrest in both the HL-60 cell line and the Colo 320HSR human colon cancer cell line. Overexpression of GSTA2 by transient transfection protected Colo 320HSR cells against both cycle arrest and apoptosis following exposure to HN2. Overexpression of GSTA2 in Colo 320HSR cells induced after exposure to HN2 did not alter cycle arrest or apoptosis. The results indicate that human GSTA2 facilitates the protection of cells from HN2 damage and not repair. Our results are consistent with the possibility that GSTA2 polymorphisms, variable isoenzyme expression, and variable induced expression may be factors in the pathogenesis of MDS.