We then explored the effect of IL-7 signaling in this system. cells, and RET expression is usually down-regulated when cytoplasmic protein is usually expressed during the pre-B cell stages of B cell development (7). E-ret transgenic mice express activated RET tyrosine kinase constitutively under the control of the E ( enhancer), driving B lineage-restricted expression of the activated RET protein. Between 3 and 8 months of life, E-ret transgenic mice develop ALL, manifested by massive adenopathy, splenomegaly, and bone marrow replacement (8-11). The malignant cells are B220+, CD43lo, and surface IgM-, and the majority are cytoplasmic – (J.F., unpublished data). Thus, the B lymphoid malignancies that arise in E-ret mice are derived from the late pro-B to early pre-B cell stage of development (12). The E-ret transgenic mouse provides a developmentally targeted model of ALL that is useful in the preclinical evaluation of novel therapeutic strategies. Cytokines play an important role in promoting and controlling normal B cell development (13, 14) and are involved in malignant transformation of lymphoid precursor cells (15). One such cytokine is usually IL-7, an important regulator of T and B cell development. IL-7 is absolutely required for normal murine T and B cell development as well as human T cell development (16). Although not absolutely required, IL-7 plays a vital role in human B cell development (17, 18). It provides a survival transmission to early B lymphoid precursors (19). When IL-7 engages the IL-7 receptor (IL-7R) on pro-B cells, IL-7R recruits intracellular kinases, resulting in cellular proliferation (20-24). In addition to its role in normal lymphoid development, IL-7 has been associated with certain malignancies (25-28). IL-7 may be associated with Hodgkin’s disease (29), Epstein-Barr virus-positive Burkitt’s lymphoma (30), and possibly T cell ALL, modulating cell cycle regulators such as p27kip1, cyclins D2 and A, CDK4, and CDK2 (27, 28). Two groups have reported that IL-7 rescues T cell ALL lymphoblasts from apoptosis (25, 26). Although there are several reports of IL-7 stimulating growth of human precursor B cell ALL cells, the role of IL-7 in the development or progression of progenitor B cell lymphoid malignancies is usually unclear (31). Rapamycin (sirolimus, Rapamune) is usually a naturally occurring immunosuppressive macrocyclic lactone that is structurally related to but biochemically unique from FK506 (tacrolimus, Prograf) (32, 33). Rapamycin inhibits the induction of activation and proliferation of mature T and B cells and is used as an immunosuppressive agent after solid organ transplant (34-38). Also, rapamycin has exhibited antitumor properties (38-40). There is evidence that mammalian target of rapamycin (mTOR) inhibitors may inhibit the growth of and/or induce apoptosis in a wide variety of tumor types (41-45). Rapamycin inhibits the activation of the mTOR. mTOR is usually a key regulator of cell growth, protein synthesis, and progression through the cell cycle (46-50). One well explained signaling intermediate in the mTOR pathway is usually p70 S6 kinase (51, 52). By inhibiting mTOR, rapamycin mimics growth-factor withdrawal, characterized by inhibition of protein synthesis and inhibition of cell cycle progression at the G1-S transition (53, 54). Although not as well studied as in T cells, rapamycin has growth-inhibitory effects in B cells (33, 35). Rapamycin inhibits secretion of soluble CD23, an autocrine B cell growth factor (55). Crosslinking of B cell receptor prospects to p70 S6 kinase Oxyclozanide activation, triggering protein synthesis by activation of ribosomal proteins (56). These data suggest that rapamycin may impact early B lineage cells, but an effect of mTOR inhibitors and IL-7-mediated signaling on early precursor B cells or on B cell progenitor malignancies has not been described. In this statement, we describe our experiments in which we evaluated the efficacy of rapamycin against progenitor B cell malignancies and the role of IL-7 in response to this mTOR inhibitor by using the E-ret transgenic mouse model. We found that (culture studies, 1-2 104 cells per well were cultured in triplicate in flat-bottom 96-well plates with increasing concentrations of rapamycin (Calbiochem) (0-100 ng/ml) and recombinant mouse IL-7 (Leinco Technologies, St. Louis) (0-30 models/ml) for 3-5 days. Cell growth was assayed by using methylthiazoletetrazolium (MTT; Sigma) as explained (57). Absorbance was measured at 595 nm by using a Benchmark microplate spectrophotometer (Bio-Rad). Results are expressed as the mean of complete absorbance of the treated sample, divided.Yada (70) have shown that IL-7 inhibits the spontaneous apoptosis of intestinal intraepithelial lymphocytes by inhibiting caspase-dependent and caspase-independent pathways. expressed during the pre-B cell stages of B cell development (7). E-ret transgenic mice express activated RET tyrosine kinase constitutively under the control of the E ( enhancer), driving B lineage-restricted expression of the activated RET protein. Between 3 and 8 months of life, E-ret transgenic mice develop ALL, manifested by massive adenopathy, splenomegaly, and bone marrow replacement (8-11). The malignant cells are B220+, CD43lo, and surface IgM-, and the majority are cytoplasmic – (J.F., unpublished data). Thus, the B lymphoid malignancies that arise in E-ret mice are derived from the late pro-B to early pre-B cell stage of development (12). The E-ret transgenic mouse provides a developmentally targeted model of ALL that is useful in the preclinical evaluation of novel therapeutic strategies. Cytokines play an important role in promoting and controlling normal B cell development (13, 14) and are involved in malignant transformation of lymphoid precursor cells (15). One such cytokine is usually IL-7, an important regulator of T and B cell development. IL-7 is absolutely required for normal murine T and B cell development as well as human T cell development (16). Although not completely required, IL-7 plays a vital role in human B cell development (17, 18). It provides a survival transmission to early B lymphoid precursors (19). When IL-7 engages the IL-7 receptor (IL-7R) on pro-B cells, IL-7R recruits intracellular kinases, resulting in cellular proliferation (20-24). In addition to its role in normal lymphoid development, IL-7 has been associated with certain malignancies (25-28). IL-7 may be associated with Hodgkin’s disease (29), Epstein-Barr virus-positive Burkitt’s lymphoma (30), and possibly T cell ALL, modulating cell cycle regulators such as p27kip1, cyclins D2 and A, CDK4, and CDK2 (27, 28). Two groups have reported that IL-7 rescues T cell ALL lymphoblasts from apoptosis (25, 26). Although there are several reports of IL-7 stimulating growth of human precursor B cell ALL cells, the role of IL-7 in the development or progression of progenitor B cell lymphoid malignancies is usually unclear (31). Rapamycin (sirolimus, Rapamune) is usually a naturally occurring immunosuppressive macrocyclic lactone that is structurally related to but biochemically unique from FK506 (tacrolimus, Prograf) (32, 33). Rapamycin inhibits the induction of activation and proliferation of mature T and B cells and is used as an immunosuppressive agent after solid organ transplant (34-38). Also, rapamycin has exhibited antitumor SOST properties (38-40). There is evidence that mammalian target of rapamycin (mTOR) inhibitors may inhibit the growth of and/or induce apoptosis in a wide variety of tumor types (41-45). Rapamycin inhibits the activation of the mTOR. mTOR is usually a key regulator of cell growth, protein synthesis, and progression through the cell cycle (46-50). One well explained signaling intermediate in the mTOR pathway is usually p70 S6 kinase (51, 52). By inhibiting mTOR, rapamycin mimics growth-factor withdrawal, characterized by inhibition of protein synthesis and inhibition Oxyclozanide of cell cycle progression at the G1-S transition (53, 54). Although not as well studied as in T cells, rapamycin has growth-inhibitory effects in B cells (33, 35). Rapamycin inhibits secretion of soluble CD23, an autocrine B cell growth factor (55). Crosslinking of B cell receptor prospects to p70 S6 kinase activation, triggering protein synthesis by activation of ribosomal proteins (56). These data suggest that rapamycin may impact early B lineage cells, but an effect of mTOR inhibitors and IL-7-mediated signaling on early precursor B cells or on B cell progenitor malignancies has not been described. In this statement, we describe our experiments in which we evaluated the efficacy of rapamycin against progenitor B cell malignancies and the role of IL-7 in response to this mTOR inhibitor by using the E-ret transgenic mouse model. We found that (culture studies, 1-2 104 cells per well were cultured in triplicate in flat-bottom 96-well plates with increasing concentrations of rapamycin (Calbiochem) (0-100 ng/ml) and recombinant mouse IL-7 (Leinco Technologies, St. Louis) (0-30 models/ml) for 3-5 days. Cell growth was assayed by using methylthiazoletetrazolium (MTT; Sigma) as explained (57). Oxyclozanide Absorbance was measured at 595 nm by using a Benchmark microplate spectrophotometer (Bio-Rad). Results are expressed as the mean of complete absorbance of the treated sample, divided by the mean of complete absorbance of the control sample. Results 1 indicate proliferation, whereas results 1 indicate growth inhibition. Apoptosis Assay. Cells (0.5-1 .