Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

Many cytokines activate two highly homologous Stat proteins, 5a and 5b. Mice deficient in both genes lack all growth hormone and prolactin functions but retain functions associated with cytokines such as erythropoietin. Here, we demonstrate that, while lymphoid development is normal, Stat5a/b mutant peripheral T cells are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression. In addition, the mice lack NK cells, develop splenomegaly, and have T cells with an activated phenotype, phenotypes seen in IL-2 receptor beta chain-deficient mice. These phenotypes are not seen in mice lacking Stat5a or Stat5b alone. The results demonstrate that the Stat5 proteins, redundantly, are essential mediators of IL-2 signaling in T cells.

ABSTRACT

Activation of Stat5 is frequently found in leukemias. To study the mechanism and role of Stat5 activation, we introduced a constitutively activated Stat5a mutant, cS5F, into murine bone marrow (BM) cells. BM transplantation with cS5F-transfected cells caused development of multilineage leukemias in lethally irradiated wild-type or nonirradiated Rag2(-/-) mice. The leukemic cells showed strongly enhanced levels of cS5F tetramers but unchanged cS5F dimer levels in a DNA binding assay. Moreover, Stat5a mutants engineered to form only dimers, but not tetramers, failed to induce leukemias. In addition, Stat5 tetramers were found to accumulate in excess compared to dimers in various human leukemias. These data suggest that Stat5 tetramers are associated with leukemogenesis.

The anti-angiogenic drug sunitinib is a receptor tyrosine-kinase inhibitor with significant, yet not curative, therapeutic impacts in metastatic renal cell carcinoma (mRCC). Sunitinib is also an immunomodulator, potently reversing myeloid-derived suppressor cell (MDSC) accumulation and T-cell inhibition in the blood even of non-responder RCC patients. We observed that sunitinib similarly prevented MDSC accumulation and restored normal T-cell function to spleens of tumor-bearing mice, independent of sunitinib's capacity to inhibit tumor progression (RENCA>CT26>4T1). Both monocytic and neutrophilic splenic MDSC were highly repressible by sunitinib. In contrast, MDSC within the microenvironment of 4T1 tumors or human RCC tumors proved highly resistant to sunitinib, and ambient T-cell function remained suppressed. Proteomic analyses comparing tumor to peripheral compartments demonstrated that GM-CSF predicted sunitinib resistance, and recombinant GM-CSF conferred sunitinib resistance to MDSC in vivo and in vitro. MDSC conditioning with GM-CSF uniquely inhibited STAT3 and promoted STAT5 activation, and STAT5ab(null/null) MDSC were rendered sensitive to sunitinib in the presence of GM-CSF in vitro. We conclude that compartment-dependent GM-CSF exposure in resistant tumors may account for sunitinib's regionalized impact upon host MDSC modulation, and hypothesize that ancillary strategies to decrease such regionalization will enhance sunitinib's potency as an immunomodulator and a cancer therapy.

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IntroductionThe functional roles of janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been characterized in both hematopoietic and nonhematopoietic tissues. 1 The JAK/STAT signaling axis allows a diverse set of extracellular signals to result in modification of gene expression patterns in the appropriate target cells. This process is initiated when, on cytokine binding, JAKs come into close proximity and become activated by autophosphorylation on tyrosine residues. Activated JAKs then phosphoryate among many other substrates, the STAT proteins. STATs form homodimers or heterodimers via SH2 domain phosphotyrosine interactions and translocate to the nucleus where they bind to transcriptional elements on DNA. 2,3 Activated STAT proteins regulate their target genes often with tissue specificity. In the hematopoietic system, JAK/STAT signaling has been extensively described in a variety of lymphoid and myeloid cell types. Four mammalian JAKs and 7 STAT proteins have been identified and characterized by generation of knockout mice. 4 These studies have revealed a large diversity in the role of STATs in hematopoiesis with some STATs showing phenotypes primarily in restricted cytokine pathways (eg, interferons, interleukin [IL]-4, IL-12, and IL-13) that are primarily involved in immune responses (STAT1, STAT2, STAT4, and STAT6). In contrast, the complex phenotypes of STAT3-and STAT5-deficient mice demonstrate a broad cytokine activation and function profile for many individual cell lineages.STAT5a and STAT5b are 2 very homologous transcription factors with variability between these 2 proteins being primarily in the transactivation domain. Mice deficient in either STAT5a or STAT5b have been generated and characterized. The STAT5a knockout mouse is characterized by defects in responses to granulocyte-macrophage colony-stimulating factor (GM-CSF) 5 and mammary gland development. 6 The STAT5b knockout mouse is characterized by defects in growth hormone signaling and expression of male-characteristic liver gene expression patterns. 7 To study the effects of STAT5 on hematopoiesis and also to eliminate any compensating function between the 2 STAT isoforms, homozygous mutant mice lacking both STAT5a and STAT5b were generated. These female mice were infertile, but heterozygous mice could breed normally and give viable STAT5ab Ϫ/Ϫ mice, but at a lower than expected frequency because of various phenotypes affecting survival. An important role for STAT5a and STAT5b in T-cell function 8,9 and terminal myeloid differentiation/apoptosis 10 has been shown by using tissues from these STAT5ab Ϫ/Ϫ mice. Comparable results have also been obtained in cytokine-inducible SH2-containing protein-1 transgenic mice. 11 Somewhat surprisingly, the peripheral hematology of the STAT5ab Ϫ/Ϫ mice was not dramatically altered, although reductions in the numbers of IL-3-and GM-CSF-responsive myeloid progenitors 12 and IL-7-responsive B-lymphoid 12,13 [25][26][27][28] have been shown to stimulate primitive hematopoi...