Endocannabinoid-like N-arachidonoyl serine is a novel pro-angiogenic mediator.

BACKGROUND AND PURPOSE: N-arachidonoyl serine (ARA-S) is a recently identified endocannabinoid-like lipid with weak affinity for the fully characterized cannabinoid receptors (CB(1) and CB(2)) and the transient receptor potential vanilloid receptor 1 (TRPV-1). ARA-S induces vasodilatation and shows vasoprotective potential via activation of key signalling pathways in endothelial cells. Based on these findings, the effect of ARA-S on endothelial functions was further studied. EXPERIMENTAL APPROACH: Primary human dermal microvascular endothelial cells (HMVEC) were used to investigate effects of ARA-S (0-10 microM) on certain endothelial functions, using cell proliferation, migration and wound repair models in vitro, and angiogenesis assays in vitro and ex vivo. Selective CB receptor antagonists and specific siRNAs were deployed to block individual CB receptors. KEY RESULTS: We found that ARA-S stimulated angiogenesis and endothelial wound healing through induction of vascular endothelial growth factor C and its cognate receptor expression in primary HMVEC. Moreover, knock-down of G protein-coupled receptor 55 (GPR55) partly inhibited ARA-S-induced signal transduction and endothelial functions. CONCLUSIONS AND IMPLICATIONS: Our results indicate that ARA-S is a pro-angiogenic factor in addition to a vessel dilator. The GPR55 receptor may serve as one target of ARA-S.

Styryl sulfonyl compounds inhibit translation of cyclin D1 in mantle cell lymphoma cells.

Mantle cell lymphoma (MCL) is characterized by the uncontrolled overexpression of cyclin D1. Styryl sulfonyl compounds have shown potent antitumor activity against MCL by inducing cell-cycle arrest and apoptosis. However, the exact molecular mechanism by which these compounds function is yet to be elucidated. Here, we show that the prototypical styryl sulfonyl compound ON 01910.Na decreased cyclin D1 and c-Myc protein levels in MCL cells, whereas mRNA levels of cyclin D1 were minimally affected. Notably, ON 01910.Na suppressed eukaryotic translation initiation factor 4E (eIF4E)-mediated cyclin D1 mRNA translation, decreased levels of phosphorylated Akt, mammalian target of Rapamycin (mTOR) and eIF4E-binding protein (eIF4E-BP), lowered the cap site binding activity of eIF4E and directly inhibited activity of phosphatidylinositol-3 kinase (PI-3K). Analysis of apoptotic signaling pathways revealed that ON 01910.Na induced the release of cytochrome c from mitochondria, altered expression of Bcl-2 family of proteins and stimulated activation of caspases. Taken together, styryl sulfonyls can cause a rapid decrease of cyclin D1 by blocking cyclin D1 mRNA translation through inhibition of the PI-3K/Akt/mTOR/eIF4E-BP signaling pathway and triggering a cytochrome c-dependent apoptotic pathway in MCL cells.

Delta9-Tetrahydrocannabinol inhibits epithelial growth factor-induced lung cancer cell migration in vitro as well as its growth and metastasis in vivo.

Delta(9)-Tetrahydrocannabinol (THC) is the primary cannabinoid of marijuana and has been shown to either potentiate or inhibit tumor growth, depending on the type of cancer and its pathogenesis. Little is known about the activity of cannabinoids like THC on epidermal growth factor receptor-overexpressing lung cancers, which are often highly aggressive and resistant to chemotherapy. In this study, we characterized the effects of THC on the EGF-induced growth and metastasis of human non-small cell lung cancer using the cell lines A549 and SW-1573 as in vitro models. We found that these cells express the cannabinoid receptors CB(1) and CB(2), known targets for THC action, and that THC inhibited EGF-induced growth, chemotaxis and chemoinvasion. Moreover, signaling studies indicated that THC may act by inhibiting the EGF-induced phosphorylation of ERK1/2, JNK1/2 and AKT. THC also induced the phosphorylation of focal adhesion kinase at tyrosine 397. Additionally, in in vivo studies in severe combined immunodeficient mice, there was significant inhibition of the subcutaneous tumor growth and lung metastasis of A549 cells in THC-treated animals as compared to vehicle-treated controls. Tumor samples from THC-treated animals revealed antiproliferative and antiangiogenic effects of THC. Our study suggests that cannabinoids like THC should be explored as novel therapeutic molecules in controlling the growth and metastasis of certain lung cancers.

Evaluation of novel cell cycle inhibitors in mantle cell lymphoma.

Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. In this study, we tested a novel class of kinase inhibitors, styryl sulfones, which differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including cyclin-dependent kinase 4, p53, mouse double minute 2 (MDM2), and cyclin D as well as increased cyclin B expression. Using both terminal deoxy transferase uridine triphosphate nick end-labelling and poly ADP-ribose polymerase assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of B-cell lymphoma family molecules. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus, styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.

Stimulation of beta 1 integrin induces tyrosine phosphorylation of vascular endothelial growth factor receptor-3 and modulates cell migration.

Interactions between integrins and tyrosine kinase receptors can modulate a variety of cell functions. We observed a cooperative interaction between the beta(1) integrin and vascular endothelial growth factor receptor-3 (VEGFR-3 or Flt4) that appeared to be required for cell migration. By using VEGFR-3-transfected 293 cells (293/VEGFR-3) or primary dermal microvascular endothelial cells (DMEC), we found that stimulation with either soluble or immobilized extracellular matrix (ECM) proteins, collagen or fibronectin (FN), resulted in the increased tyrosine phosphorylation of VEGFR-3 in the absence of a cognate ligand. This increased tyrosine phosphorylation of VEGFR-3 was diminished by pretreatment with a blocking antibody against the beta(1) integrin. Cross-linking with anti-beta(1) integrin antibody induced a similar degree of tyrosine phosphorylation of VEGFR-3. Stimulation with collagen or FN induced an association between beta(1) integrin and VEGFR-3 in both 293/VEGFR-3 and primary DMEC cells. Collagen or FN-induced tyrosine phosphorylation of VEGFR-3 was inhibited by treatment with cytochalasin D, an inhibitor of actin polymerization. Collagen or FN was able to induce the migration of 293/VEGFR-3 or DMEC cells to a limited extent. However, migration was dramatically enhanced when a gradient of the cognate ligand, VEGF-D, was added. VEGF-D failed to induce cell migration in the absence of ECM proteins. Introducing a mutation at the kinase domain of VEGFR-3 or treatment with blocking antibody against either VEGFR-3 or beta(1) integrin inhibited cell migration induced by ECM and VEGF-D, indicating that signals from both beta(1) integrin and VEGFR-3 are required for this cell function.

HIV-1 Tat induces microvascular endothelial apoptosis through caspase activation.

HIV-1 Tat, in addition to its critical role in viral transcription, is secreted from infected cells and can act as a proto-cytokine. We studied the effects of HIV-1 Tat in primary human microvascular endothelial cells of lung origin and found that it caused apoptosis. This apoptosis occurred without induction of either Fas or TNF, known mediators of programmed cell death. Tat, like Fas ligand, induced cleavage of chromatin structure, as evidenced by changes in DNA laddering, incorporation of fluorescein into the nicked chromosomal DNA (TUNEL assay), and mono- or oligonucleosomes. Furthermore, Tat treatment caused cleavage of poly(A/DP)-ribose polymerase, a substrate of caspases. Caspase-3, but not caspase-9, was activated following treatment of primary human microvascular endothelial cells of lung origin with either Tat or anti-Fas agonist Ab (anti-Fas). Inhibition of caspase-3 activity markedly reduced apoptosis. Although Fas-mediated apoptosis involved changes in Bcl-2, Bax, and Bad regulatory proteins, such alterations were not observed with Tat. Taken together, these data demonstrate that HIV-1 Tat is able to activate apoptosis in microvascular endothelium by a mechanism distinct from TNF secretion or the Fas pathway.

Beta-chemokine induction of activation protein-1 and cyclic AMP responsive element activation in human myeloid cells.

Chemokines effect leukocyte chemotaxis and activation through their binding to specific G protein-coupled receptors. Although early steps in chemokine signal transduction pathways have been characterized, there is relatively limited information available at the transcription factor level. To that end, we have examined the binding activity on activation protein-1 (AP-1) and cyclic AMP responsive element (CRE) target sequences in human THP-1 myeloid cells after treatment with the beta-chemokine macrophage inflammatory protein (MIP-1alpha). MIP-1alpha induced both AP-1 and CRE activation. Although inhibition of protein kinase C blocked the AP-1 activity induced by this chemokine, there was no decrease in CRE activation in the presence of a protein kinase A inhibitor. Using kinase assays, it appeared that mitogen-activated protein kinase pathways were involved in CRE activation. In addition, HIV-1 infection of THP-1 cells resulted in constitutive activation of AP-1 and CRE elements but no further response to MIP-1alpha treatment. These results suggest that beta-chemokines act via protein kinase C-dependent pathways and mitogen-activated protein kinase pathways to modulate the host transcriptional response in myeloid cells, and that this response is altered by HIV infection.

Janus kinase 2 is involved in stromal cell-derived factor-1alpha-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells.

Stromal cell-derived factor-1 (SDF-1), the ligand for the CXCR4 receptor, is a highly efficacious chemoattractant for CD34(+) hematopoietic progenitor cells. However, the SDF-1/CXCR4 signaling pathways that regulate hematopoiesis are still not well defined. This study reports that SDF-1alpha can stimulate the tyrosine phosphorylation of Janus kinase 2 (JAK2) and other members of the JAK/signal transduction and activation of transcription (STAT) family, including JAK1, tyrosine kinase 2, STAT2, and STAT4 in the human progenitor cell line, CTS. SDF-1alpha stimulation of these cells also enhanced the association of JAK2 with phosphatidylinositol 3 (PI3)-kinase. This enhanced association was abolished by pretreatment of cells with AG490, a specific JAK2 inhibitor. Furthermore, pretreatment of CTS cells with AG490 significantly inhibited SDF-1alpha-induced PI3-kinase activity, and inhibition of JAK2 with AG490 ablated the SDF-1alpha-induced tyrosine phosphorylation of multiple focal adhesion proteins (including focal adhesion kinase, related adhesion focal tyrosine kinase, paxillin, CrkII, CrkL, and p130Cas). Chemotaxis assays showed that inhibition of JAK2 diminished SDF-1alpha-induced migration in both CTS cells and CD34(+) human bone marrow progenitor cells. Hence, these results suggest that JAK2 is required for CXCR4 receptor-mediated signaling that regulates cytoskeletal proteins and cell migration through PI3-kinase pathways in hematopoietic progenitor cells. (Blood. 2001;97:3342-3348)

HIV-1 Tat promotes monocyte chemoattractant protein-1 secretion followed by transmigration of monocytes.

The mechanism whereby HIV-infected cells transit from the bloodstream into tissues is not well defined. This phenomenon was addressed by studying the effects of HIV-1 Tat, a protein secreted by infected cells, on human lung microvascular endothelial cells (HMVEC-Ls). It was found that monocyte chemoattractant protein-1 (MCP-1) was released from HMVEC-Ls in a dose- and time-dependent manner after Tat treatment. MCP-1 is a potent beta-chemokine that recruits monocytes and T cells and promotes cell adhesion and transmigration across an endothelial monolayer. It was also observed that MCP-1 and the culture medium from Tat-treated HMVEC-Ls were chemotactic for CD14(+) monocytes from human peripheral blood and for THP-1, a promonocytic cell line used as a model system. To characterize the signaling pathways underlying the observed induction of MCP-1, HMVEC-Ls were treated with 2 different protein kinase inhibitors: PD98059, a MAP kinase inhibitor, and GF109203X, a protein kinase C (PKC) inhibitor. MCP-1 release was significantly reduced when PKC was inhibited, and slightly decreased when PI3 kinase was blocked; no effect on MCP-1 release was observed on MAP kinase inhibition. Similarly, transmigration of THP-1 cells was significantly impaired by the PKC inhibitor, but not by the other tested inhibitors. These data indicate that the HIV-1 Tat protein may act as a protocytokine by causing the release of MCP-1 from the endothelial monolayer, and thereby facilitating monocyte transmigration into tissues via a PKC signaling pathway.

Impact of cancer-related fatigue on the lives of patients: new findings from the Fatigue Coalition.

PURPOSE: This survey was designed to confirm the prevalence and duration of fatigue in the cancer population and to assess its physical, mental, social, and economic impacts on the lives of patients and caregivers. Patients and Methods. A 25-minute telephone interview was completed with 379 cancer patients having a prior history of chemotherapy. Patients were recruited from a sample of 6, 125 households in the United States identified as having a member with cancer. The median patient age was 62 years, and 79% of respondents were women. Patients reporting fatigue at least a few times a month were asked a series of questions to better describe their fatigue and its impact on quality of life. RESULTS: Seventy-six percent of patients experienced fatigue at least a few days each month during their most recent chemotherapy; 30% experienced fatigue on a daily basis. Ninety-one percent of those who experienced fatigue reported that it prevented a "normal" life, and 88% indicated that fatigue caused an alteration in their daily routine. Fatigue made it more difficult to participate in social activities and perform typical cognitive tasks. Of the 177 patients who were employed, 75% changed their employment status as a result of fatigue. Furthermore, 65% of patients indicated that their fatigue resulted in their caregivers taking at least one day (mean, 4.5 days) off work in a typical month. Physicians were the health care professionals most commonly consulted (79%) to discuss fatigue. Bed rest/ relaxation was the most common treatment recommendation (37%); 40% of patients were not offered any recommendations. CONCLUSIONS: Cancer-related fatigue is common among cancer patients who have received chemotherapy and results in substantial adverse physical, psychosocial, and economic consequences for both patients and caregivers. Given the impact of fatigue, treatment options should be routinely considered in the care of patients with cancer.

HIV-1 gp120- and gp160-induced apoptosis in cultured endothelial cells is mediated by caspases.

The immune dysfunction and cell destruction that occur in the human immunodeficiency virus (HIV)-infected host appear to result from the direct cytopathic effects of viral infection and the effects of viral proteins on uninfected bystander cells. Recently, the alpha-chemokine receptor CXCR4 has been reported to mediate apoptosis in neuronal cells and in CD4(+) and CD8(+) T cells after its binding to HIV-1 envelope proteins. In the current study, it was observed that human umbilical vein endothelial cells (HUVEC) undergo apoptosis after their treatment with the HIV-1 envelope proteins gp120/160. Anti-CXCR4 monoclonal antibody decreased HIV-1 gp120/160-induced apoptosis, suggesting that the CXCR4 chemokine receptor mediates the apoptotic effects of these HIV envelope glycoproteins. Further studies revealed that caspases play an important role in this process because the pretreatment of cells with a general caspase enzyme inhibitor decreased the extent of HUVEC apoptosis induced by gp120/160. In addition, it was found that caspase-3 was activated on HIV-1 gp120/160 treatment of these cells. It was also observed that gp120/160 treatment slightly increased the expression of the pro-apoptotic molecule Bax. These results suggest that HIV-1 envelope glycoproteins can disrupt endothelial integrity through the interaction with CXCR4, thereby facilitating virus transit out of the bloodstream and contributing to the vascular injury syndromes seen in acquired immunodeficiency syndrome. (Blood. 2000;96:1438-1442)

Beta-chemokine receptor CCR5 signals through SHP1, SHP2, and Syk.

The beta-chemokine receptor CCR5 has been shown to modulate cell migration, proliferation, and immune functions and to serve as a co-receptor for the human immunodeficiency virus. We and others have shown that CCR5 activates related adhesion focal tyrosine kinase (RAFTK)/Pyk2/CAK-beta. In this study, we further characterize the signaling molecules activated by CCR5 upon binding to its cognate ligand, macrophage inflammatory protein-1beta (MIP1beta). We observed enhanced tyrosine phosphorylation of the phosphatases SHP1 and SHP2 upon MIP1beta stimulation of CCR5 L1.2 transfectants and T-cells derived from peripheral blood mononuclear cells. Furthermore, we observed that SHP1 associated with RAFTK. However, using a dominant-negative phosphatase-binding mutant of RAFTK (RAFTK(m906)), we found that RAFTK does not mediate SHP1 or SHP2 phosphorylation. SHP1 and SHP2 also associated with the adaptor protein Grb2 and the Src-related kinase Syk. Pretreatment of CCR5 L1.2 transfectants or T-cells with the phosphatase inhibitor orthovanadate markedly abolished MIP1beta-induced chemotaxis. Syk was also activated upon MIP1beta stimulation of CCR5 L1.2 transfectants or T-cells and associated with RAFTK. Overexpression of a dominant-negative Src-binding mutant of RAFTK (RAFTK(m402)) significantly attenuated Syk activation, whereas overexpression of wild-type RAFTK enhanced Syk activity, indicating that RAFTK acts upstream of CCR5-mediated Syk activation. Taken together, these results suggest that MIP1beta stimulation mediated by CCR5 induces the formation of a signaling complex consisting of RAFTK, Syk, SHP1, and Grb2.

Chemokine receptors CXCR-1/2 activate mitogen-activated protein kinase via the epidermal growth factor receptor in ovarian cancer cells.

Ovarian cancer typically disseminates widely in the abdomen, a characteristic that limits curative therapy. The mechanisms that promote ovarian cancer cell migration are incompletely understood. We studied model SK-OV-3 ovarian cancer cells and observed robust expression of the alpha chemokine receptors CXCR-1 and CXCR-2. Interleukin-8 (IL-8) treatment caused shape changes in the cells, with membrane ruffling and formation/retraction of thin actin-like projections, as detected by time-lapse microscopy. Stimulation of the CXCR-1/2 receptors by human interleukin 8 (IL-8) rapidly activated the p44/42 mitogen-activated protein (extracellular signal-regulated kinase (Erk1/2)) kinase pathway. Treatment of SK-OV-3 cells with the inhibitors genestein and herbimycin A indicated that tyrosine kinases were involved in the IL-8 activation of Erk1 and Erk2. Of note, IL-8 induced transient phosphorylation of the epidermal growth factor (EGF) receptor and its association with the adaptor molecules Shc and Grb2. This transactivation of the EGF receptor was dependent on intracellular Ca(2+) mobilization. Furthermore AG1478, a specific inhibitor of the EGF receptor kinase, blocked Erk1 and Erk2 activation. c-Src kinase was not involved in the IL-8-mediated phosphorylation of the EGF receptor, but was critical for Shc phosphorylation and downstream Erk1/2 kinase activation. These results suggest important "cross-talk" between chemokine and growth factor pathways that may link signals of cell migration and proliferation in ovarian cancer.

c-Src mediates mitogenic signals and associates with cytoskeletal proteins upon vascular endothelial growth factor stimulation in Kaposi's sarcoma cells.

Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi's sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.

Stromal cell-derived factor-1alpha stimulates tyrosine phosphorylation of multiple focal adhesion proteins and induces migration of hematopoietic progenitor cells: roles of phosphoinositide-3 kinase and protein kinase C.

The stromal cell-derived factor-1 (SDF-1) is an alpha chemokine that binds to the CXCR4 receptor. Knock-out studies in mice demonstrate that this ligand-receptor pair is essential in hematopoiesis. One function of SDF-1 appears to be the regulation of migration of hematopoietic progenitor cells. We previously characterized signal transduction pathways induced by SDF-1alpha in human hematopoietic progenitors and found tyrosine phosphorylation of focal adhesion components, including the related adhesion focal tyrosine kinase (RAFTK), the adaptor molecule p130 Cas, and the cytoskeletal protein paxillin. To better understand the functional role of signaling molecules connecting the CXCR4 receptor to the process of hematopoietic migration, we studied SDF-1alpha-mediated pathways in a model hematopoietic progenitor cell line (CTS), as well as in primary human bone marrow CD34(+) cells. We observed that several other focal adhesion components, including focal adhesion kinase (FAK) and the adaptor molecules Crk and Crk-L, are phosphorylated on SDF-1alpha stimulation. Using a series of specific small molecule inhibitors, both protein kinase C (PKC) and phosphoinositide-3 kinase (PI-3K) appeared to be required for SDF-1alpha-mediated phosphorylation of focal adhesion proteins and the migration of both CTS and primary marrow CD34(+) cells, whereas the mitogen-activated protein kinases ERK-1 and -2 were not. These studies further delineate the molecular pathways mediating hematopoietic progenitor migration and response to an essential chemokine, SDF-1alpha. (Blood. 2000;95:2505-2513)

Expression and utilization of co-receptors in HIV and simian immunodeficiency virus infection of megakaryocytes.

OBJECTIVE: To analyse the expression and specificity of co-receptors for the entry of HIV and simian immunodeficiency virus (SIV) into megakaryocytes. DESIGN AND METHODS: The expression of co-receptors was determined by flow cytometric analysis in combination with reverse transcription-polymerase chain (RT-PCR) reaction. The specificity of co-receptors in virus entry was determined by the infection of HIV-1 pseudotyped with X4- (HXB2), R5- (YU2), or R5X4-tropic (89.6) envelope proteins of HIV-1 or with envelope proteins of SIVpbj1.9. RESULTS: The model human erythroleukemia (HEL) cell line, exhibiting megakaryocytic-like properties, expressed CCR5, CCR3, CXCR4, and CPR15/BOB, and all viruses except YU2 (R5) efficiently entered the cells. The blocking of virus entry with specific chemokines showed that the entry of HXB2 (X4) was impaired by SDF-1beta but not by other chemokines, indicating that CXCR4 was a major co-receptor for the entry of HXB2. Primary human bone marrow megakaryocytes displayed a different repertoire of co-receptor expression from that of HEL cells, as all viruses except YU2 efficiently entered these cells. However, chemokine blocking experiments showed that the entry of HXB2 into primary bone marrow megakaryocytes was insufficiently blocked by SDF-1beta compared with the entry into HEL cells, suggesting that alternative co-receptors could be employed for the entry of X4 virus into bone marrow cells. CONCLUSION: These data suggest that cells of megakaryocytic lineage are susceptible to infection by X4 viruses, with less marked susceptibility to R5 isolates, and that SDF-1beta efficiently blocks the infection of HEL cells but not of primary bone marrow megakaryocytes. Our data reveal that novel co-receptors are probably utilized for the entry of X4 virus into megakaryocytes.

Kaposi's sarcoma-associated herpesvirus-encoded G protein-coupled receptor activation of c-jun amino-terminal kinase/stress-activated protein kinase and lyn kinase is mediated by related adhesion focal tyrosine kinase/proline-rich tyrosine kinase 2.

The Kaposi's sarcoma-associated herpesvirus (KSHV) (also known as human herpesvirus 8) has been implicated in the pathogenesis of Kaposi's sarcoma and B cell primary effusion lymphomas. KSHV encodes a G protein-coupled receptor (GPCR) that acts as an oncogene and constitutively activates two protein kinases, c-Jun amino-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase. It also induces the production of vascular endothelial growth factor. These processes are believed to be important in KSHV-GPCR-related oncogenesis. We have characterized the signaling pathways mediated by KSHV-GPCR in a reconstituted 293T cell model in which the related adhesion focal tyrosine kinase (RAFTK) was ectopically expressed. RAFTK has been shown to play an important role in growth factor signaling in endothelium and in B cell antigen receptor signaling in B lymphocytes. KSHV-GPCR induced the tyrosine phosphorylation of RAFTK. Expression of wild-type RAFTK enhanced GPCR-mediated JNK/SAPK activation, whereas dominant-negative mutant constructs of RAFTK, such as K457A (which lacks kinase activity) and Y402F (a Src-binding mutant), inhibited KSHV-GPCR-mediated activation of JNK/SAPK. RAFTK also mediated the KSHV-GPCR-induced activation of Lyn, a Src family kinase. However, RAFTK did not mediate the activation of p38 mitogen-activated protein kinase induced by KSHV-GPCR. Human interferon gamma-inducible protein-10, which is known to inhibit KSHV-GPCR activity, was found to reduce RAFTK phosphorylation and JNK/SAPK activation. These results suggest that in cells expressing RAFTK/proline-rich tyrosine kinase 2, such as endothelial and B cells, RAFTK can act to enhance KSHV-GPCR-mediated downstream signaling to transcriptional regulators such as JNK/SAPK.

Chemotherapy-induced anemia in adults: incidence and treatment.

Anemia is a common complication of myelosuppressive chemotherapy that results in a decreased functional capacity and quality of life (QOL) for cancer patients. Severe anemia is treated with red blood cell transfusions, but mild-to-moderate anemia in patients receiving chemotherapy has traditionally been managed conservatively on the basis of the perception that it was clinically unimportant. This practice has been reflected in the relative inattention to standardized and complete reporting of all degrees of chemotherapy-induced anemia. We undertook a comprehensive review of published chemotherapy trials of the most common single agents and combination chemotherapy regimens, including the new generation of chemotherapeutic agents, used in the treatment of the major nonmyeloid malignancies in adults to characterize and to document the incidence and severity of chemotherapy-induced anemia. Despite identified limitations in the grading and reporting of treatment-related anemia, the results confirm a relatively high incidence of mild-to-moderate anemia. Recent advances in assessing the relationships of anemia, fatigue, and QOL in cancer patients are providing new insights into these closely related factors. Clinical data are emerging that suggest that mild-to-moderate chemotherapy-induced anemia results in a perceptible reduction in a patient's energy level and QOL. Future research may lead to new classifications of chemotherapy-induced anemia that can guide therapeutic interventions on the basis of outcomes and hemoglobin levels. Perceptions by oncologists and patients that lesser degrees of anemia must be endured without treatment may be overcome as greater emphasis is placed on the QOL of the oncology patient and as research provides further insights into the relationships between hemoglobin levels, patient well-being, and symptoms.

CD4 receptor-dependent entry of human immunodeficiency virus type-1 env-pseudotypes into CCR5-, CCR3-, and CXCR4-expressing human alveolar macrophages is preferentially mediated by the CCR5 coreceptor.

Alveolar macrophages (AM) are important host-defense cells and targets of human immunodeficiency virus type 1 (HIV-1) infection. However, the receptors mediating HIV-1 entry into AM are not completely characterized. We observed that, in addition to CD4 receptors, AM from healthy adults expressed low levels of CCR5, CCR3, and CXCR4 chemokine receptors by flow cytometry, and specific messenger RNA was detected for all three receptors by reverse transcriptase/polymerase chain reaction. The macrophage monocytotropic (M-tropic; YU2) and dual-tropic (89.6) HIV-1 env-pseudotypes entered AM efficiently, as expected given CCR3 and CCR5 expression. However, the T-lymphocytotropic (T-tropic; HXB2) pseudotype did not enter AM despite expression of the appropriate chemokine coreceptor CXCR4. Incubation of AM with regulated on activation, normal T cells expressed and secreted (RANTES) significantly impaired entry of the M-tropic (YU2) HIV-1 pseudotype, whereas SDF-1beta or eotaxin did not impair entry. The entry of simian immunodeficiency virus (SIV) pbj1.9 env-pseudotype into AM was not blocked by RANTES, SDF-1beta, or eotaxin. The competence of these chemokine receptors for virus entry was confirmed in Cf2Th canine thymocytes cotransfected with the human CD4 and chemokine receptors. Entry of the M-tropic (YU2) HIV-1 pseudotype was shown to be mediated by either CCR3 or CCR5, the T-tropic (HXB2) HIV-1 pseudotype by CXCR4, and the dual-tropic (89.6) HIV-1 or the SIVpbj1. 9 pseudotype by CCR5, CCR3, or CXCR4. Our data indicate that the mechanisms for HIV-1 entry are both receptor-specific and cell type-specific, and that chemokine receptor expression on AM does not fully explain cell susceptibility to different virus isolates.

Stromal cell-derived factor-1 alpha and stem cell factor/kit ligand share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis.

Stromal cell-derived factor (SDF-1alpha), the ligand for CXCR4, is a chemokine that acts as a potent chemoattractant for hemopoietic progenitor cells. Stem cell factor/kit ligand (SCF/KL), an early acting cytokine, has recently been reported to enhance the chemotaxis induced by SDF-1alpha. However, very little is known about downstream signaling events following these receptor-ligand interactions. To investigate these events, we utilized a model progenitor cell line, CTS, which expresses both the CXCR4 and c-kit receptors. We observed strong Ca2+ mobilization and enhancement of chemotaxis following treatment with SDF-1alpha or SCF/KL. A combination of these factors enhanced this chemotaxis in CTS cells as well as in CD34+ bone marrow cells. Prior treatment of CTS cells with pertussis toxin inhibited the SDF-1alpha-induced chemotaxis, suggesting that SDF-1alpha signaling involves a pertussis-sensitive Gi-coupled protein. SDF-1alpha treatment resulted in a rapid phosphorylation of the focal adhesion molecules RAFTK (related adhesion focal tyrosine kinase), paxillin, and p130cas, which then declined within minutes. SCF/KL alone or in combination with SDF-1alpha induced a rapid and sustained effect on phosphorylation of these substrates. SDF-1alpha treatment resulted in a rapid and robust activation of p44/42 mitogen-activated protein kinase compared with the relatively weak and delayed effect of SCF/KL treatment. Interestingly, a delayed but sustained activation of mitogen-activated protein kinase activation was observed when the factors were used in combination. Such cooperativity in downstream signaling pathways may explain the enhanced chemotaxis of progenitors observed with SDF-1alpha in combination with SCF/KL.