Abstract
Abstract 1695
Introduction:
The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples.
Methods:
The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained.
Results:
A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18).
Conclusions:
Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy.
Disclosures:
No relevant conflicts of interest to declare.
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