IntroductionB-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of a monoclonal population of CD5 ϩ neoplastic B cells in secondary lymphoid organs, marrow, and blood. Because most of the circulating leukemia cells are arrested in the G 0 /G 1 phase of the cell cycle, the primary defect may be one of resistance to programmed cell death rather than accelerated cell division. 1,2 However, CLL cells can rapidly undergo spontaneous apoptosis under culture conditions that support the growth of human B-cell lines. This implies that such ex vivo conditions lack factors necessary for leukemia cell survival or that the resistance to apoptosis is not intrinsic to the leukemia B cell.The leukemia cell microenvironment in the marrow or in secondary lymphoid tissues may contribute to the noted resistance of CLL cells to apoptosis in vivo. 3,4 Normal B-cell development depends on complex interactions with accessory cells that define the so-called specialized microenvironments. T cells and a variety of different types of adherent cells, generally defined as stromal cells, are the main elements of the microenvironment. 3 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the pattern and extent of marrow involvement correlates with clinical stage and prognosis. 5,6 As such, interactions with stromal cells in the marrow microenvironment appear to play a role in disease progression and resistance to therapy. [7][8][9] In addition, we found that a small proportion of the mononuclear cells from the blood of patients with CLL can differentiate into large, round, adherent cells that attract CLL cells and protect them from undergoing spontaneous or drug-induced cell death. 10,11 Because these cells share features with thymic nurse cells that nurture developing thymocytes, we designated them nurselike cells (NLCs). Although NLCs differentiate from blood mononuclear cells after several days in vitro, fully differentiated NLCs can be found in the spleen and secondary lymphoid tissue of patients with CLL, 11 where they might play a role in protecting CLL cells from apoptosis in vivo. This model implies that CLL cells depend on specific extrinsic factors from NLCs and other stromal elements for their survival. Conceivably, CLL cells recirculate from the blood through secondary lymphoid tissues and back into the systemic circulation in response to certain chemokines.One such chemokine is stromal cell-derived factor-1/pre-B cell growth-stimulating factor (SDF-1/PBSF), which recently has been designated CXCL12. CXCL12 is a member of a family of chemotactic cytokines (chemokines) that initially were characterized as growth-stimulating factors for B-cell precursors. 12 CXCR4 is a primary physiologic receptor for CXCL12 and functions as a coreceptor for entry of T-tropic strains of HIV-1. Mutant mice with targeted gene disruption of CXCL12 or CXCR4 have defects in the We previously demonstrated that stromal cells can attract CLL cells through the production of CXCL12. 13 In addition, NLCs e...

Small-cell lung cancer (SCLC) is an aggressive, rapidly metastasizing neoplasm. The chemokine stromal cellderived factor-1 (SDF-1/CXCL12) is constitutively secreted by marrow stromal cells and plays a key role for homing of hematopoietic cells to the marrow. Here, we report that tumor cells from patients with SCLC express high levels of functional CXCR4 receptors for the chemokine CXCL12. Reverse transcriptase-polymerase chain reaction and flow cytometry demonstrated CXCR4 mRNA and CXCR4 surface expression in SCLC cell lines. Immunohistochemistry of primary tumor samples from SCLC patients revealed high expression of CXCR4. CXCL12 elicited CXCR4 receptor endocytosis, actin polymerization, and a robust activation of phospho-p44/ 42 mitogen-activated protein kinase in SCLC cells. Furthermore, CXCL12 induced SCLC cell invasion into extracellular matrix and firm adhesion to marrow stromal cells. Stromal cell adhesion of SCLC cells was significantly inhibited by the specific CXCR4 antagonist T140, pertussis toxin, antivascular cell adhesion molecule-1(VCAM-1) antibodies, and CS-1 peptide, demonstrating the importance of CXCR4 chemokine receptor activation and a4b1 integrin binding, respectively. In addition, CXCL12 enhanced the adhesion of SCLC cells to immobilized VCAM-1, demonstrating that CXCR4 chemokine receptors can induce integrin activation on SCLC cells. As SCLC has a high propensity for bone marrow involvement, our findings suggest that CXCR4 chemokine receptors and a4b1 integrins play a critical role in the interaction of SCLC cells with stromal cells in the tumor microenvironment.

Small cell lung cancer (SCLC) is an aggressive, rapidly metastazising neoplasm with a high propensity for marrow involvement. SCLC cells express high levels of functional CXCR4 receptors for the chemokine stromal-cell-derived factor-1 (SDF-1/CXCL12). Adhesion of SCLC cells to extracellular matrix or accessory cells within the tumor microenvironment confers resistance to chemotherapy via integrin signaling and thus may be responsible for residual disease and relapses commonly seen in SCLC. We examined the signaling mechanisms that regulate CXCL12-induced adhesion of SCLC cells to fibronectin, collagen, and stromal cells and the effects on SCLC cell chemoresistance. We found that CXCL12-induced integrin activation which resulted in an increased adhesion of SCLC cells to fibronectin and collagen. This was mediated by a2, a4, a5, and b1 integrins along with CXCR4 activation, which could be inhibited by CXCR4 antagonists. Stromal cells protected SCLC cells from chemotherapy-induced apoptosis, and this protection could also be antagonized by CXCR4 inhibitors. We conclude that activation of integrins and CXCR4 chemokine receptors co-operate in mediating adhesion and survival signals from the tumor microenvironment to SCLC cells. Therefore, CXCR4 antagonists in combination with cytotoxic drugs should be explored in SCLC to overcome CXCL12-mediated adhesion and survival signals in the tumor microenvironment.

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The Hedgehog (Hh) pathway regulates cell proliferation and survival and contributes to tumorigenesis. We investigated the expression and function of this pathway in B-cell chronic lymphocytic leukemia (CLL) cells and in healthy B lymphocytes. Profiling of cognate Hh pathway members revealed reduced expression of two key Hh signaling effectors, Smoothened (SMOH) and GLI, in CLL cells, whereas transcription levels of other investigated members resembled normal B-lymphocyte levels. Examining the functional role of SMOH and GLI in cell survival, we found that CLL cells were hardly sensitive toward specific SMOH inhibition, but showed an unspecific decline in cell viability in response to high concentrations of the SMOH antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT61 reduced expression of the target gene Patched and preferentially decreased the viability of malignant cells. Specific RNA interference knockdown experiments in a CLL-derived cell line confirmed the autonomous role of GLI in malignant cell survival. GANT61-induced apoptosis in primary leukemic cells was partly attenuated by protective stromal cells, but not soluble sonic hedgehog ligand. In summary, our data show a downregulation of the classical Hh pathway in CLL and suggest an intrinsic SMOH-independent role of GLI in the ex vivo survival of CLL cells.

SummaryThe human chemokine receptor CRAM (chemokine receptor on activated macrophages), encoded by the gene CCRL2, is a new candidate for the atypical chemokine receptor family that includes the receptors DARC, D6 and chemocentryx chemokine receptor (CCX-CKR). CRAM is maturation-stage-dependently expressed on human B lymphocytes and its surface expression is up-regulated upon short-term CCL5 exposure. Here, we demonstrate that the homeostatic chemokine CCL19 is a specific ligand for CRAM. In radioactive labelling studies CCL19 bound to CRAMexpressing cells with an affinity similar to the described binding of its other receptor CCR7. In contrast to the known CCL19/CCR7 ligand/ receptor pair, CRAM stimulation by CCL19 did not result in typical chemokine-receptor-dependent cellular activation like calcium mobilization or migration. Instead, we demonstrate that CRAM is constitutively recycling via clathrin-coated pits and able to internalize CCL19 as well as anti-CRAM antibodies. As this absence of classical chemokine receptor responses and the recycling and internalization features are characteristic for non-classical chemokine receptors, we suggest that CRAM is the newest member of this group. As CCL19 is known to be critically involved in lymphocyte and dendritic cell trafficking, CCL19-binding competition by CRAM might be involved in modulating these processes.

Key Points Motility of resting CLL cells requires chemokine-mediated RhoA activation but is independent of Tiam1/Rac signals. Tiam1/Rac signals are indispensible for CLL cell proliferation and chemoresistance.

The Kaposi's sarcoma herpesvirus encodes a G-proteincoupled chemokine receptor termed KSHV-GPCR. Expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi's sarcoma. Previously, we have shown that CXCR2, the closest human homolog, is similarly able to transform cells if continuously stimulated or constitutively activated by amino-acid exchange D138V of the DRY sequence. Here, we demonstrate that STAT3 activation is a prerequisite for transformation in KSHV-GPCR and CXCR2 transfected NIH 3T3 cells. In KSHV-GPCR and D138V transfected cells, STAT-3 is constitutively phosphorylated on Tyr 705 . In CXCR2 transfected NIH 3T3 cells and human microvascular endothelial cells (HMEC), which express the CXCR2 constitutively, STAT3 is phosphorylated upon stimulation with IL-8 (CXCL8). Focus formation in NIH 3T3 cells transfected with the KSHV-GPCR, CXCR2, or the D138V mutant, was blocked by the specific JAK2 inhibitor AG490. Typical functions of the CXCR2 including actin stress fiber formation, haptotaxis, and the angiogenic response in HMEC shown by tube formation in Matrigel were blocked by AG490. These data suggest that the transforming capacity and migratory responses that are involved in tumor development, metastasis, and angiogenesis in KSHV or CXCR2-expressing cells is at least partially mediated through a JAK2-STAT3 dependent pathway.