Tyrosine kinase inhibitors (TKIs) are promising new agents for specific inhibition of malignant cell growth and metastasis formation. Because most of the TKIs have to reach an intracellular target, specific membrane transporters may significantly modulate their effectiveness. In addition, the hydrophobic TKIs may interact with so-called multidrug transporters and thus alter the cellular distribution of unrelated pharmacological agents. In the present work, we show that certain TKIs, already in the clinical phase of drug development, directly interact with the ABCG2 multidrug transporter protein with a high affinity. We found that in several in vitro assay systems, STI-571 (Gleevec; imatinib mesylate), ZD1839 (Iressa; gefitinib), and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (EKI-785) interacted with ABCG2 at submicromolar concentrations, whereas other multidrug transporters, human multidrug resistance protein (P-glycoprotein, ABCB1) and human multidrug resistance protein 1 (ABCC1), showed much lower reactivity toward these agents. Low concentrations of the TKIs examined selectively modulated ABCG2-ATPase activity, inhibited ABCG2-dependent active drug extrusion, and significantly affected drug resistance patterns in cells expressing ABCG2. Our results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the clinical treatment of cancer patients. These data also raise the possibility that an extrusion of TKIs by multidrug transporters, e.g., ABCG2, may be involved in tumor cell TKI resistance.

Iressa (ZD1839, Gefitinib), used in clinics to treat non-small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signalingdependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa. (Cancer Res 2005; 65(5): 1770-7)

The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function.

Ring1 and Yy1 Binding Protein (Rybp) has been implicated in transcriptional regulation, apoptotic signaling and as a member of the polycomb repressive complex 1 has important function in regulating pluripotency and differentiation of embryonic stem cells. Earlier, we have proven that Rybp plays essential role in mouse embryonic and central nervous system development. This work identifies Rybp, as a critical regulator of heart development. Rybp is readily detectable in the developing mouse heart from day 8.5 of embryonic development.Prominent Rybp expression persists during all embryonic stages and Rybp marks differentiated cell types of the heart. By utilizing rybp null embryonic stem cells (ESCs) in an in vitro cardiac differentiation assay we found that rybp null ESCs do not form rhythmically beating cardiomyocytes. Gene expression profiles revealed a down-regulation of terminal cardiac and upregulation of germ line specific markers in the rybp null cardiomyocytes. Furthermore, transcriptome analysis uncovered a number of novel candidate target genes regulated by Rybp.Among these are several important in cardiac development and contractility such as Plagl1, Isl1, This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. 2Tnnt2. Importantly, forced expression of rybp in rybp deficient ESCs by a lentiviral vector was able to rescue the mutant phenotype. Our data provides evidence for a previously unrecognized function of Rybp in heart development.and pointing out the importance of germ cell lineage gene silencing during somatic differentiation. IntroductionA complex network of transcription factors governs the temporal and spatial patterns of gene expression in the organs of the developing embryo proper. Transcription factors also have important roles in postnatal and adult life in maintaining the pattern of differentiated gene expression [1][2][3]. Ring1 and Yy1 binding protein (Rybp; or also known as Dedaf (Death Effector Domain Associated Factor), UniGene Mm.321633; MGI:1929059) is a repressor protein that is also a member of the mammalian polycomb repressive complex 1 (PRC1) [4]. Rybp first was described as binding partner for the polycomb group protein (PcG) Ring1A (Ring1; ortholog of Drosophila dRing/Sce) and also was shown to associate with Ring1B (Ring2/Rnf2; ortholog of Drosophila dRing/Sce) and M33 (Pc1; ortholog of Drosophila Pc), components of the PRC1 multiprotein complex [5]. Our previous work showed that Rybp is selectively upregulated in distinct structures and cell types of the developing eye and the central nervous system (CNS) and, may also play a role in the development of more mature neurons [6,7]. We have also shown that rybp is essential for the development of the mouse embryo proper and that homozygous null mouse embryos cannot develop further implantation (embryonic day 5.5 (E5.5)). Notably, in a subset of heterozygous animals and in rybp −/− ↔rybp +/+ chimeras, alt...

Cell proliferation and differentiation are highly coordinated processes during normal development. Most leukemia cells are blocked from undergoing terminal differentiation and also exhibit uncontrolled proliferation. Dysregulated expression of transcription factor PU.1 is strongly associated with Friend virus-induced erythroleukemia. PU.1 inhibits erythroid differentiation by binding to and inhibiting GATA-1. PU.1 also may be involved in controlling proliferation of erythroid cells. We reported previously that the G 1 phase-specific cyclin-dependent kinase 6 (CDK6) also blocks erythroid differentiation. We now report that PU.1 directly stimulates transcription of the cdk6 gene in both normal erythroid progenitors and erythroleukemia cells, as well as in macrophages. We propose that PU.1 coordinates proliferation and differentiation in immature erythroid cells by inhibiting the GATA-1-mediated gene expression program and also by regulating expression of genes that control progression through the G 1 phase of the cell cycle, the period during which the decision to differentiate is made.