Restoring Cell Responsiveness to GM-CSF and IL-3 by PTEN Is Via Controlling the Regulation of Egr-1 In TF-1a Cell Line

Abstract 2504

Cytokine independence is a common event in leukemia. The mechanism is often linked to deregulation of Ras/PI3K, Ras/MAP, and JAK/STAT pathways. PTEN, a major negative regulator of the Ras pathway, was initially isolated as a tumor suppressor in a variety of malignancies. We recently reported that PTEN is deficient in 67% of juvenile myelomonocytic leukemia (JMML) patients. Other groups have also found alterations of PTEN, PI3K, or AKT in 47.7% of T-cell acute lymphoblastic leukemia. Mice who are rendered pten-deficient develop myeloproliferative disorders and acute myeloid and lymphoid leukemias, and the leukemia-initiating stem cell in these mouse models could be distinguished from the normal hematopoietic stem cell by a differential response to rapamycin. The transcription factor, Egr-1, has been known to be a positive regulator of PTEN expression. It plays an important role in controlling proliferation as well as mobilization of hematopoietic stem cells. It is unknown whether PTEN had a feedback loop on regulating Egr-1 expression. Since poorly differentiated progenitors are often mobilized to the peripheral blood in leukemia patients, we hypothesized that PTEN deficiency alters the feedback loop of Egr-1 in response to cytokine stimulation. In this manner, PTEN deficiency might contribute to the cytokine independence observed in many types of leukemia. In order to evaluate this hypothesis we investigated the role of PTEN in cells responding to GM-CSF stimulation in the leukemia cell line, TF-1a. TF-1a is PTEN deficient as well as cytokine independent, and displays a constitutively high expression of Egr-1. We restored PTEN expression in TF-1a cells. This was accomplished by packaging the coding sequence of wild-type PTEN in a retrovirus vector, pBMN-GFP, that carried a gene encoding green fluorescent protein (GFP). TF-1a cells were transfected with pBMN-PTEN-GFP or pBMN-GFP by spinoculation. The pure population of transfected cells was sorted by FACS, followed by limiting dilution. The transfected single colonies were expanded for further evaluation of PTEN expression and biological function. Data from these experiments showed: 1) over-expression PTEN in TF-1a down-regulated the expression of Egr-1 and restored the responsiveness of Egr-1 to GM-CSF stimulation, 2) PTEN restored the responsiveness of TF-1a to GM-CSF and IL-3, and 3) these events are CREB-independent. In conclusion, our data demonstrates that PTEN contributes to the cell responsiveness to GM-CSF and IL-3, and has a negative feedback role to regulate Egr-1 expression in TF-1a cells. In addition to Egr-1 regulating PTEN in a positive manner, our data indicates the presence of a feedback loop from PTEN back to Egr-1. This appears to be an event independent of CREB.