Fumin Chang - Academia.edu (original) (raw)

Papers by Fumin Chang

Leukemia, 2000

The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine r... more The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in beta-estradiol-inducible constructs. We introduced these chimeric deltaRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cytokine-dependent cells that expressed the deltaRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the deltaRaf:ER oncoprotein. Depending upon the particular deltaRaf:ER oncoprotein, cytokine-dependent cells were recovered 10(3) to 10(5) times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the deltaRaf:ER o...

Effects of deregulated RAF and MEK1 expression on the cytokine-dependency of hematopoietic cells

Advances in Enzyme Regulation, 2000

... Exp. Med. [1980]. Raf-1 protein is required for growth factor-induced proliferation of hemato... more ... Exp. Med. [1980]. Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells. (PMID:7539043) Muszynski KW, Ruscetti FW, Heidecker G, Rapp U, Troppmair J, Gooya JM, Keller JR. J. Exp. Med. [1995 ...

Leukemia, 2001

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated... more The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using ⌬MEK1:ER, a conditionally active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine dependency of the murine lymphoid hematopoietic cell line FL5.12. Cytokine-independent cells were obtained from FL5.12 cells at a frequency of 1 × 10 −7 , indicating that a low frequency of cells expressing ⌬MEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to ⌬MEK1:ER activation than those that remained cytokine-dependent. ⌬MEK1:ER-responsive cells could be maintained long-term in the presence of ␤estradiol, as well as the estrogen-receptor antagonist 4-hydroxy-tamoxifen. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that activated ⌬MEK1:ER may induce a pathway leading to autocrine proliferation. Cytokine-dependent ⌬MEK1:ER cells were found to increase the expression of GM-CSF receptor ␣ (GM-CSFR␣) in response to ␤-estradiol. In contrast, MEK1responsive cells were found to express constitutively lower levels of GM-CSFR␣ and ␤ common (␤ c ) chains indicating that constitutive GM-CSF expression resulted in a decrease in GM-CSFR expression. Treatment of parental cells with supernatant from MEK1-responsive FL5.12 cells was sufficient to promote [ 3 H]-thymidine incorporation. GM-CSF was found to enhance the viability of FL5.12 cells. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells. Leukemia (2001) 15, 794-807.

International Journal of Cancer, 2002

Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-ca... more Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-carotene lowers the risk of cancer, recent intervention studies have revealed that ␤-carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that ␤-carotene may act as a pro-or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide-differentiated HL-60 cells exposed to ␤-carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose-dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, ␤-carotene was more effective in decreasing cyclin A and Bcl-2 expression and in increasing p21 and p27 expression. Neither Bcl-xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by ␤-carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, ␣-tocopherol minimized the effects of ␤-carotene on cell growth. These data provide evidence that ␤-carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells.

Leukemia, 1999

Over the past decade, there has been an exponential increase in our knowledge of how cytokines re... more Over the past decade, there has been an exponential increase in our knowledge of how cytokines regulate signal transduction, cell cycle progression, differentiation and apoptosis. Research has focused on different biochemical and genetic aspects of these processes. Initially, cytokines were identified by clonogenic assays and purified by biochemical techniques. This soon led to the molecular cloning of the genes encoding the cytokines and their cognate receptors. Determining the structure and regulation of these genes in normal and malignant hematopoietic cells has furthered our understanding of neoplastic transformation. Furthermore, this has allowed the design of modified cytokines which are able to stimulate multiple receptors and be more effective in stimulating the repopulation of hematopoietic cells after myelosuppressive chemotherapy. The mechanisms by which cytokines transduce their regulatory signals have been evaluated by identifying the involvement of specific protein kinase cascades and their downstream transcription factor targets. The effects of cytokines on cell cycle regulatory molecules, which either promote or arrest cell cycle progression, have been more recently examined. In addition, the mechanisms by which cytokines regulate apoptotic proteins, which mediate survival vs death, are being elucidated. Identification and characterization of these complex, interconnected pathways has expanded our knowledge of leukemogenesis substantially. This information has the potential to guide the development of therapeutic drugs designed to target key intermediates in these pathways and effectively treat patients with leukemias and lymphomas. This review focuses on the current understanding of how hematopoietic cytokines such as IL-3, as well as its cognate receptor, are expressed and the mechanisms by which they transmit their growth regulatory signals. The effects of aberrant regulation of these molecules on signal transduction, cell cycle regulatory and apoptotic pathways in transformed hematopoietic cells are discussed. Finally, anti-neoplastic drugs that target crucial constituents in these pathways are evaluated.

Advances in Enzyme Regulation, 2001

Interactions between the PI3K and Raf signaling pathways can result in the transformation of hematopoietic cells

Cancer detection and prevention, 2001

The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals fro... more The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals from membrane receptors to downstream targets that regulate apoptosis, gene expression, and cell growth. The abilities of activated PI3K, Akt, Raf, and MEK proteins to abrogate the cytokine dependence of three different hematopoietic cell lines were determined. Activated PI3K or Akt expression by themselves did not efficiently annul cytokine dependence. Raf and MEK could abrogate the cytokine dependence of murine FDC-PI and human TF-1 cells; however, the frequency of transformation was dependent on the particular oncogene examined, as more factor-independent cells were isolated after infection with activated retroviruses encoding A-Raf or Raf-1 than were with MEK1 or B-Raf. Cytokine-independent deltaRaf-1-infected cells formed tumors on injection into immunocompromised mice, whereas cytokine-dependent cell lines did not, demonstrating the oncogenic effects of activation of the Raf/MEK/ERK p...

Leukemia, 2000

The MEK1 oncoprotein plays a critical role in Ras/Raf/ MEK/MAPK-mediated transmission of mitogeni... more The MEK1 oncoprotein plays a critical role in Ras/Raf/ MEK/MAPK-mediated transmission of mitogenic signals from cell surface receptors to the nucleus. In order to examine this pathway's role in leukemic transformation, a conditionally active (␤-estradiol-inducible) form of the MEK1 protein was created by ligating a cDNA encoding an N-terminal truncated form of MEK1 to the hormone-binding domain of the estrogen receptor (ER). We introduced this chimeric ⌬MEK1:ER oncoprotein into cytokine-dependent human TF-1 and murine FDC-P1 hematopoietic cell lines. Two different types of cells were recovered after drug selection in medium containing either cytokine or ␤estradiol: (1) cells that expressed the ⌬MEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to ⌬MEK1:ER activation. Cytokinedependent cells were recovered 10 2 to 10 4 times more frequently than MEK1-responsive cells depending upon the particular cell line. To determine whether BCL2 overexpression could synergize with the ⌬MEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent ⌬MEK1:ERexpressing cells were infected with a BCL2-containing retrovirus, and the frequency of MEK1-responsive cells determined. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cells, however, it did increase the frequency at which MEK1-responsive cells were recovered approximately 10-fold. ⌬MEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of ␤-estradiol in the MEK1responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to ⌬MEK1:ER stimulation in both ⌬MEK1:ER and ⌬MEK1:ER+BCL2 cells. As compared to the cytokine-dependent ⌬MEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive ⌬MEK1:ER and ⌬MEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive ⌬MEK1:ER+BCL2 cells than in the MEK1-responsive cells lacking BCL2 or cytokine-dependent cells. These conditionally transformed cells will be useful in furthering our understanding of the roles MEK1 and BCL2 play in the prevention of apoptosis in hematopoietic cells. Leukemia (2000) 14, 1080-1096.

Pharmacology & Therapeutics, 2000

This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellula... more This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signal transduction pathway and the consequences of its unregulation in the development of cancer. The roles of some of the cell membrane receptors involved in the activation of this pathway, the G-protein Ras, the Raf, MEK and ERK kinases, the phosphatases that regulate these kinases, as well as the downstream transcription factors that become activated, are discussed. The roles of the Ras ± Raf ± MEK ± ERK pathway in the regulation of apoptosis and cell cycle progression are also analyzed. In addition, potential targets for pharmacological intervention in growth factor-responsive cells are evaluated. D 2001 Elsevier Science Inc. All rights reserved.

Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention

Leukemia, 2003

The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kina... more The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade couples signals from cell surface receptors to transcription factors, which regulate gene expression. Depending upon the stimulus and cell type, this pathway can transmit signals, which result in the prevention or induction of apoptosis or cell cycle progression. Thus, it is an appropriate pathway to target for therapeutic intervention. This pathway becomes more complex daily, as there are multiple members of the kinase and transcription factor families, which can be activated or inactivated by protein phosphorylation. The diversity of signals transduced by this pathway is increased, as different family members heterodimerize to transmit different signals. Furthermore, additional signal transduction pathways interact with the Raf/MEK/ERK pathway to regulate positively or negatively its activity, or to alter the phosphorylation status of downstream targets. Abnormal activation of this pathway occurs in leukemia because of mutations at Ras as well as genes in other pathways (eg PI3K, PTEN, Akt), which serve to regulate its activity. Dysregulation of this pathway can result in autocrine transformation of hematopoietic cells since cytokine genes such as interleukin-3 and granulocyte/macrophage colony-stimulating factor contain the transacting binding sites for the transcription factors regulated by this pathway. Inhibitors of Ras, Raf, MEK and some downstream targets have been developed and many are currently in clinical trials. This review will summarize our current understanding of the Ras/Raf/MEK/ERK signal transduction pathway and the downstream transcription factors. The prospects of targeting this pathway for therapeutic intervention in leukemia and other cancers will be evaluated.

Critical Roles of the Raf/MEK/ERK Pathway in Apoptosis and Drug Resistance

Apoptosis, Cell Signaling, and Human Diseases, 2007

... James A. McCubrey, Fred E. Bertrand, Linda S. Steelman, Fumin Chang, David M. Terrian, and Ri... more ... James A. McCubrey, Fred E. Bertrand, Linda S. Steelman, Fumin Chang, David M. Terrian, and Richard A. Franklin ... Raf activity (shown by a black P in a white circle) whereas others serve to inhibit Raf activity (shown by a white P in a black circle. ...

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Journal of Histochemistry & Cytochemistry, 2014

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vit... more Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.

Circulation Research, 2014

Rationale: Increased arginase activity contributes to endothelial dysfunction by competition for ... more Rationale: Increased arginase activity contributes to endothelial dysfunction by competition for l-arginine substrate and reciprocal regulation of nitric oxide synthase (NOS). The rapid increase in arginase activity in human aortic endothelial cells exposed to oxidized low-density lipoprotein (OxLDL) is consistent with post-translational modification or subcellular trafficking.

Novel targets for reversing endothelial dysfunction in atherogenesis: Mitochondrial processing peptidase and HDAC2

Nitric Oxide, 2014

Pharmacology & Therapeutics, 2000

This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellula... more This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signal transduction pathway and the consequences of its unregulation in the development of cancer. The roles of some of the cell membrane receptors involved in the activation of this pathway, the G-protein Ras, the Raf, MEK and ERK kinases, the phosphatases that regulate these kinases, as well as the downstream transcription factors that become activated, are discussed. The roles of the Ras ± Raf ± MEK ± ERK pathway in the regulation of apoptosis and cell cycle progression are also analyzed. In addition, potential targets for pharmacological intervention in growth factor-responsive cells are evaluated. D 2001 Elsevier Science Inc. All rights reserved.

P21Cip1 induced by Raf is associated with increased Cdk4 activity in hematopoietic cells

Oncogene, 2001

To investigate the functions of the different Raf genes in hematopoietic cell proliferation, the ... more To investigate the functions of the different Raf genes in hematopoietic cell proliferation, the capacities of beta-estradiol-regulated Delta Raf:ER genes to induce cell cycle regulatory gene expression and cell cycle progression in FDC-P1 cells were examined. Raf activation increased the expression of Cdk2, Cdk4, cyclin A, cyclin D, cyclin E, p21(Cip1) and c-Myc and decreased the expression of p27(Kip1) which are associated with G(1) progression. However only the cell clones with moderate Raf activation, i.e. FD/Delta Raf-1:ER and FD/Delta A-Raf:ER, successfully underwent cell proliferation. The cell clones with the highest Delta Raf activity, FD/Delta B-Raf:ER, underwent apoptosis before cell proliferation. p21(Cip1) induced by Raf activation specifically bound with Cdk4/cyclin D complexes but not Cdk2/cyclin E complexes and this binding was associated with the increased Cdk4 activity. However, no binding of p27(Kip1) with either Cdk2/cyclin E or Cdk4/cyclin D was observed. Thus Raf mediated growth was associated with elevated p21(Cip1) expression, which may specifically bind with and activate Cdk4/cyclin D complexes and with decreased p27(Kip1) expression.

Molecular Biology of the Cell, 2006

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon ce... more FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosinephosphorylated ␤PIX and thereby increased its binding to Rac1. In addition, ␤PIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of ␤PIX.

International Journal of Cancer, 2002

Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-ca... more Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-carotene lowers the risk of cancer, recent intervention studies have revealed that ␤-carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that ␤-carotene may act as a pro-or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide-differentiated HL-60 cells exposed to ␤-carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose-dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, ␤-carotene was more effective in decreasing cyclin A and Bcl-2 expression and in increasing p21 and p27 expression. Neither Bcl-xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by ␤-carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, ␣-tocopherol minimized the effects of ␤-carotene on cell growth. These data provide evidence that ␤-carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells.

Glycobiology, 1997

To establish a model system for the study of ganglioside metabolism of the human brain tumor, med... more To establish a model system for the study of ganglioside metabolism of the human brain tumor, medulloblastoma, we have chemically characterized the gangliosides of the Daoy cell line. These cells contain a high concentration of gangliosides (143 ± 13 nmol LBSA/10 8 cells). The major species have been structurally confirmed to be G M2 (65.9%), G^ (13.0%), and G Dla (103%). Isolation of individual gangliosides homogeneous in both carbohydrate and ceramide moieties by reversed-phase HPLC and analysis by negative-ion fast atom bombardment collisionally activated dissociation tandem mass spectrometry have allowed us to unequivocally characterize ceramide structures. In the case of G^, 10 major ceramide subspecies were identified: dl8:l-hC16:0, dl8:l-C16:0, dl8:0-C16:0, dl8:l-C18:0, dl8:l-C20:0, dl8:l-C22:0, dl8:2-C24:l, dl8: 1-C23:1, dl8:l-C24:l, and dl8:l-C24:0. Taken together with previous studies, these findings in human medulloblastoma cells support the view that high expression and marked heterogeneity of ceramide structure are general characteristics of tumor gangliosides, molecules which are shed by the tumor cells and which are biologically active in vivo.

Experimental Cell Research, 1997

Among the tumor -host interactions believed to fa-Shedding of immunosuppressive gangliosides is a... more Among the tumor -host interactions believed to fa-Shedding of immunosuppressive gangliosides is an vor tumor cell survival, one process is the release or important characteristic of both experimental and hu-shedding of tumor cell surface molecules which may man tumors. Using a medulloblastoma cell line, Daoy, have important biological properties . We are studywith a very high ganglioside expression (141 { 13 nmol/ ing a specific class of cell surface glycosphingolipids, 10 8 cells) and a well-characterized ganglioside complegangliosides, in this context. Gangliosides consist of a ment, we have now studied ganglioside shedding by sialic acid-containing carbohydrate portion and a lipid human brain tumor cells. Shedding of gangliosides, portion (ceramide) embedded in the cell membrane. quantified by metabolic radiolabeling, was significant Ganglioside molecules have a number of important bi-(169 pmol/10 8 cells/h) and was generalized with respect ological properties which could conceivably into the major ganglioside carbohydrate structures (G M2 , fluence the survival of the tumor cells which carry G M3 , and G D1a ). For each ganglioside, however, shedthese molecules. They have been shown to be immunoding was selective for ceramide structures containing suppressive [10-12], to enhance tumor formation in shorter fatty acyl chains. Rapid and ceramide-selecvivo , and to enhance platelet activation . Tutive shedding was confirmed in two additional human mor gangliosides have also been shown to have angiomedulloblastoma cell lines, D341 Med and D283 Med genic activity , which could promote neovascu-(112 and 59 pmol/10 8 cells/h). Significant ganglioside larization of a tumor bed. shedding is therefore a common characteristic of hu-Given the above biologically significant activities, the man medulloblastoma cells and may influence the bioultimate relevance of tumor gangliosides to tumor forlogical behavior of this tumor, in view of immunosupmation nevertheless depends on their access or availpressive and other biological properties of shed gangliability to ''sites of action.'' In other words, in order to osides. ᭧ 1997 Academic Press

Leukemia, 2000

The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine r... more The Raf oncoprotein plays critical roles in the transmission of mitogenic signals from cytokine receptors to the nucleus. There are three Raf family members: A-Raf, B-Raf and Raf-1. Conditionally active forms of the Raf proteins were created by ligating N-terminal truncated activated forms to the estrogen-receptor (ER) hormone-binding domain resulting in beta-estradiol-inducible constructs. We introduced these chimeric deltaRaf:ER oncoproteins into the murine FDC-P1 hematopoietic cell line. Two different types of cells were recovered after drug selection in medium containing either cytokine or beta-estradiol: (1) cytokine-dependent cells that expressed the deltaRaf:ER oncoproteins; and (2) Raf-responsive cells that grew in response to the deltaRaf:ER oncoprotein. Depending upon the particular deltaRaf:ER oncoprotein, cytokine-dependent cells were recovered 10(3) to 10(5) times more frequently than Raf-responsive cells. To determine whether BCL2 could synergize with the deltaRaf:ER o...

Effects of deregulated RAF and MEK1 expression on the cytokine-dependency of hematopoietic cells

Advances in Enzyme Regulation, 2000

... Exp. Med. [1980]. Raf-1 protein is required for growth factor-induced proliferation of hemato... more ... Exp. Med. [1980]. Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells. (PMID:7539043) Muszynski KW, Ruscetti FW, Heidecker G, Rapp U, Troppmair J, Gooya JM, Keller JR. J. Exp. Med. [1995 ...

Leukemia, 2001

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated... more The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using ⌬MEK1:ER, a conditionally active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine dependency of the murine lymphoid hematopoietic cell line FL5.12. Cytokine-independent cells were obtained from FL5.12 cells at a frequency of 1 × 10 −7 , indicating that a low frequency of cells expressing ⌬MEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to ⌬MEK1:ER activation than those that remained cytokine-dependent. ⌬MEK1:ER-responsive cells could be maintained long-term in the presence of ␤estradiol, as well as the estrogen-receptor antagonist 4-hydroxy-tamoxifen. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that activated ⌬MEK1:ER may induce a pathway leading to autocrine proliferation. Cytokine-dependent ⌬MEK1:ER cells were found to increase the expression of GM-CSF receptor ␣ (GM-CSFR␣) in response to ␤-estradiol. In contrast, MEK1responsive cells were found to express constitutively lower levels of GM-CSFR␣ and ␤ common (␤ c ) chains indicating that constitutive GM-CSF expression resulted in a decrease in GM-CSFR expression. Treatment of parental cells with supernatant from MEK1-responsive FL5.12 cells was sufficient to promote [ 3 H]-thymidine incorporation. GM-CSF was found to enhance the viability of FL5.12 cells. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells. Leukemia (2001) 15, 794-807.

International Journal of Cancer, 2002

Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-ca... more Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-carotene lowers the risk of cancer, recent intervention studies have revealed that ␤-carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that ␤-carotene may act as a pro-or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide-differentiated HL-60 cells exposed to ␤-carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose-dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, ␤-carotene was more effective in decreasing cyclin A and Bcl-2 expression and in increasing p21 and p27 expression. Neither Bcl-xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by ␤-carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, ␣-tocopherol minimized the effects of ␤-carotene on cell growth. These data provide evidence that ␤-carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells.

Leukemia, 1999

Over the past decade, there has been an exponential increase in our knowledge of how cytokines re... more Over the past decade, there has been an exponential increase in our knowledge of how cytokines regulate signal transduction, cell cycle progression, differentiation and apoptosis. Research has focused on different biochemical and genetic aspects of these processes. Initially, cytokines were identified by clonogenic assays and purified by biochemical techniques. This soon led to the molecular cloning of the genes encoding the cytokines and their cognate receptors. Determining the structure and regulation of these genes in normal and malignant hematopoietic cells has furthered our understanding of neoplastic transformation. Furthermore, this has allowed the design of modified cytokines which are able to stimulate multiple receptors and be more effective in stimulating the repopulation of hematopoietic cells after myelosuppressive chemotherapy. The mechanisms by which cytokines transduce their regulatory signals have been evaluated by identifying the involvement of specific protein kinase cascades and their downstream transcription factor targets. The effects of cytokines on cell cycle regulatory molecules, which either promote or arrest cell cycle progression, have been more recently examined. In addition, the mechanisms by which cytokines regulate apoptotic proteins, which mediate survival vs death, are being elucidated. Identification and characterization of these complex, interconnected pathways has expanded our knowledge of leukemogenesis substantially. This information has the potential to guide the development of therapeutic drugs designed to target key intermediates in these pathways and effectively treat patients with leukemias and lymphomas. This review focuses on the current understanding of how hematopoietic cytokines such as IL-3, as well as its cognate receptor, are expressed and the mechanisms by which they transmit their growth regulatory signals. The effects of aberrant regulation of these molecules on signal transduction, cell cycle regulatory and apoptotic pathways in transformed hematopoietic cells are discussed. Finally, anti-neoplastic drugs that target crucial constituents in these pathways are evaluated.

Advances in Enzyme Regulation, 2001

Interactions between the PI3K and Raf signaling pathways can result in the transformation of hematopoietic cells

Cancer detection and prevention, 2001

The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals fro... more The PI3K/Akt and Raf/MEK/ERK signal transduction cascades are pivotal in transmitting signals from membrane receptors to downstream targets that regulate apoptosis, gene expression, and cell growth. The abilities of activated PI3K, Akt, Raf, and MEK proteins to abrogate the cytokine dependence of three different hematopoietic cell lines were determined. Activated PI3K or Akt expression by themselves did not efficiently annul cytokine dependence. Raf and MEK could abrogate the cytokine dependence of murine FDC-PI and human TF-1 cells; however, the frequency of transformation was dependent on the particular oncogene examined, as more factor-independent cells were isolated after infection with activated retroviruses encoding A-Raf or Raf-1 than were with MEK1 or B-Raf. Cytokine-independent deltaRaf-1-infected cells formed tumors on injection into immunocompromised mice, whereas cytokine-dependent cell lines did not, demonstrating the oncogenic effects of activation of the Raf/MEK/ERK p...

Leukemia, 2000

The MEK1 oncoprotein plays a critical role in Ras/Raf/ MEK/MAPK-mediated transmission of mitogeni... more The MEK1 oncoprotein plays a critical role in Ras/Raf/ MEK/MAPK-mediated transmission of mitogenic signals from cell surface receptors to the nucleus. In order to examine this pathway's role in leukemic transformation, a conditionally active (␤-estradiol-inducible) form of the MEK1 protein was created by ligating a cDNA encoding an N-terminal truncated form of MEK1 to the hormone-binding domain of the estrogen receptor (ER). We introduced this chimeric ⌬MEK1:ER oncoprotein into cytokine-dependent human TF-1 and murine FDC-P1 hematopoietic cell lines. Two different types of cells were recovered after drug selection in medium containing either cytokine or ␤estradiol: (1) cells that expressed the ⌬MEK1:ER oncoprotein but remained cytokine-dependent and (2) MEK1-responsive cells that grew in response to ⌬MEK1:ER activation. Cytokinedependent cells were recovered 10 2 to 10 4 times more frequently than MEK1-responsive cells depending upon the particular cell line. To determine whether BCL2 overexpression could synergize with the ⌬MEK1:ER oncoprotein in relieving cytokine dependence, the cytokine-dependent ⌬MEK1:ERexpressing cells were infected with a BCL2-containing retrovirus, and the frequency of MEK1-responsive cells determined. BCL2 overexpression, by itself, did not relieve cytokine dependency of the parental cells, however, it did increase the frequency at which MEK1-responsive cells were recovered approximately 10-fold. ⌬MEK1:ER+BCL2 cells remained viable for at least 3 days after estradiol deprivation, whereas viability was readily lost upon withdrawal of ␤-estradiol in the MEK1responsive cells which lacked BCL2 overexpression. The MAP kinases, ERK1 and ERK2 were activated in response to ⌬MEK1:ER stimulation in both ⌬MEK1:ER and ⌬MEK1:ER+BCL2 cells. As compared to the cytokine-dependent ⌬MEK1:ER and BCL2 infected cells, MEK1-responsive BCL2 infected cells expressed higher levels of BCL2. While both MEK1-responsive ⌬MEK1:ER and ⌬MEK1:ER+BCL2 infected cells expressed cDNAs encoding the autocrine cytokine GM-CSF, more GM-CSF cDNAs and bioactivity were detected in the MEK1-responsive ⌬MEK1:ER+BCL2 cells than in the MEK1-responsive cells lacking BCL2 or cytokine-dependent cells. These conditionally transformed cells will be useful in furthering our understanding of the roles MEK1 and BCL2 play in the prevention of apoptosis in hematopoietic cells. Leukemia (2000) 14, 1080-1096.

Pharmacology & Therapeutics, 2000

This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellula... more This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signal transduction pathway and the consequences of its unregulation in the development of cancer. The roles of some of the cell membrane receptors involved in the activation of this pathway, the G-protein Ras, the Raf, MEK and ERK kinases, the phosphatases that regulate these kinases, as well as the downstream transcription factors that become activated, are discussed. The roles of the Ras ± Raf ± MEK ± ERK pathway in the regulation of apoptosis and cell cycle progression are also analyzed. In addition, potential targets for pharmacological intervention in growth factor-responsive cells are evaluated. D 2001 Elsevier Science Inc. All rights reserved.

Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention

Leukemia, 2003

The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kina... more The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade couples signals from cell surface receptors to transcription factors, which regulate gene expression. Depending upon the stimulus and cell type, this pathway can transmit signals, which result in the prevention or induction of apoptosis or cell cycle progression. Thus, it is an appropriate pathway to target for therapeutic intervention. This pathway becomes more complex daily, as there are multiple members of the kinase and transcription factor families, which can be activated or inactivated by protein phosphorylation. The diversity of signals transduced by this pathway is increased, as different family members heterodimerize to transmit different signals. Furthermore, additional signal transduction pathways interact with the Raf/MEK/ERK pathway to regulate positively or negatively its activity, or to alter the phosphorylation status of downstream targets. Abnormal activation of this pathway occurs in leukemia because of mutations at Ras as well as genes in other pathways (eg PI3K, PTEN, Akt), which serve to regulate its activity. Dysregulation of this pathway can result in autocrine transformation of hematopoietic cells since cytokine genes such as interleukin-3 and granulocyte/macrophage colony-stimulating factor contain the transacting binding sites for the transcription factors regulated by this pathway. Inhibitors of Ras, Raf, MEK and some downstream targets have been developed and many are currently in clinical trials. This review will summarize our current understanding of the Ras/Raf/MEK/ERK signal transduction pathway and the downstream transcription factors. The prospects of targeting this pathway for therapeutic intervention in leukemia and other cancers will be evaluated.

Critical Roles of the Raf/MEK/ERK Pathway in Apoptosis and Drug Resistance

Apoptosis, Cell Signaling, and Human Diseases, 2007

... James A. McCubrey, Fred E. Bertrand, Linda S. Steelman, Fumin Chang, David M. Terrian, and Ri... more ... James A. McCubrey, Fred E. Bertrand, Linda S. Steelman, Fumin Chang, David M. Terrian, and Richard A. Franklin ... Raf activity (shown by a black P in a white circle) whereas others serve to inhibit Raf activity (shown by a white P in a black circle. ...

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Journal of Histochemistry & Cytochemistry, 2014

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vit... more Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.

Circulation Research, 2014

Rationale: Increased arginase activity contributes to endothelial dysfunction by competition for ... more Rationale: Increased arginase activity contributes to endothelial dysfunction by competition for l-arginine substrate and reciprocal regulation of nitric oxide synthase (NOS). The rapid increase in arginase activity in human aortic endothelial cells exposed to oxidized low-density lipoprotein (OxLDL) is consistent with post-translational modification or subcellular trafficking.

Novel targets for reversing endothelial dysfunction in atherogenesis: Mitochondrial processing peptidase and HDAC2

Nitric Oxide, 2014

Pharmacology & Therapeutics, 2000

This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellula... more This review focuses on the Ras ± Raf ± mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signal transduction pathway and the consequences of its unregulation in the development of cancer. The roles of some of the cell membrane receptors involved in the activation of this pathway, the G-protein Ras, the Raf, MEK and ERK kinases, the phosphatases that regulate these kinases, as well as the downstream transcription factors that become activated, are discussed. The roles of the Ras ± Raf ± MEK ± ERK pathway in the regulation of apoptosis and cell cycle progression are also analyzed. In addition, potential targets for pharmacological intervention in growth factor-responsive cells are evaluated. D 2001 Elsevier Science Inc. All rights reserved.

P21Cip1 induced by Raf is associated with increased Cdk4 activity in hematopoietic cells

Oncogene, 2001

To investigate the functions of the different Raf genes in hematopoietic cell proliferation, the ... more To investigate the functions of the different Raf genes in hematopoietic cell proliferation, the capacities of beta-estradiol-regulated Delta Raf:ER genes to induce cell cycle regulatory gene expression and cell cycle progression in FDC-P1 cells were examined. Raf activation increased the expression of Cdk2, Cdk4, cyclin A, cyclin D, cyclin E, p21(Cip1) and c-Myc and decreased the expression of p27(Kip1) which are associated with G(1) progression. However only the cell clones with moderate Raf activation, i.e. FD/Delta Raf-1:ER and FD/Delta A-Raf:ER, successfully underwent cell proliferation. The cell clones with the highest Delta Raf activity, FD/Delta B-Raf:ER, underwent apoptosis before cell proliferation. p21(Cip1) induced by Raf activation specifically bound with Cdk4/cyclin D complexes but not Cdk2/cyclin E complexes and this binding was associated with the increased Cdk4 activity. However, no binding of p27(Kip1) with either Cdk2/cyclin E or Cdk4/cyclin D was observed. Thus Raf mediated growth was associated with elevated p21(Cip1) expression, which may specifically bind with and activate Cdk4/cyclin D complexes and with decreased p27(Kip1) expression.

Molecular Biology of the Cell, 2006

FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon ce... more FAK, a cytoplasmic protein tyrosine kinase, is activated and localized to focal adhesions upon cell attachment to extracellular matrix. FAK null cells spread poorly and exhibit altered focal adhesion turnover. Rac1 is a member of the Rho-family GTPases that promotes membrane ruffling, leading edge extension, and cell spreading. We investigated the activation and subcellular location of Rac1 in FAK null and FAK reexpressing fibroblasts. FAK reexpressers had a more robust pattern of Rac1 activation after cell adhesion to fibronectin than the FAK null cells. Translocation of Rac1 to focal adhesions was observed in FAK reexpressers, but seldom in FAK null cells. Experiments with constitutively active L61Rac1 and dominant negative N17Rac1 indicated that the activation state of Rac1 regulated its localization to focal adhesions. We demonstrated that FAK tyrosinephosphorylated ␤PIX and thereby increased its binding to Rac1. In addition, ␤PIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of ␤PIX.

International Journal of Cancer, 2002

Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-ca... more Although epidemiologic studies have demonstrated that a high intake of vegetables containing ␤-carotene lowers the risk of cancer, recent intervention studies have revealed that ␤-carotene supplementation to smokers resulted in a high incidence of lung cancer. We hypothesized that ␤-carotene may act as a pro-or anticancerogenic agent by modulating pathways involved in cell growth and that such a modulation may involve a redox mechanism. To test this hypothesis, cell proliferation, apoptosis and redox status were evaluated in undifferentiated and dimethylsulfoxide-differentiated HL-60 cells exposed to ␤-carotene. The carotenoid modified cell cycle progression and induced apoptosis in a dose-dependent manner. These effects were more remarkable in undifferentiated cells than in differentiated cells. In accord with these findings, in undifferentiated cells, ␤-carotene was more effective in decreasing cyclin A and Bcl-2 expression and in increasing p21 and p27 expression. Neither Bcl-xL nor Bax expression were significantly modified by the carotenoid. From a mechanistic point of view, the delay in cell growth by ␤-carotene was highly coincident with the increased intracellular reactive oxygen species production and oxidized glutathione content induced by the carotenoid. Moreover, ␣-tocopherol minimized the effects of ␤-carotene on cell growth. These data provide evidence that ␤-carotene modulates molecular pathways involved in cell cycle progression and apoptosis and support the hypothesis that a redox mechanism may be implicated. They also suggest that differentiated cells may be less susceptible to the carotenoid than highly neoplastic undifferentiated cells.

Glycobiology, 1997

To establish a model system for the study of ganglioside metabolism of the human brain tumor, med... more To establish a model system for the study of ganglioside metabolism of the human brain tumor, medulloblastoma, we have chemically characterized the gangliosides of the Daoy cell line. These cells contain a high concentration of gangliosides (143 ± 13 nmol LBSA/10 8 cells). The major species have been structurally confirmed to be G M2 (65.9%), G^ (13.0%), and G Dla (103%). Isolation of individual gangliosides homogeneous in both carbohydrate and ceramide moieties by reversed-phase HPLC and analysis by negative-ion fast atom bombardment collisionally activated dissociation tandem mass spectrometry have allowed us to unequivocally characterize ceramide structures. In the case of G^, 10 major ceramide subspecies were identified: dl8:l-hC16:0, dl8:l-C16:0, dl8:0-C16:0, dl8:l-C18:0, dl8:l-C20:0, dl8:l-C22:0, dl8:2-C24:l, dl8: 1-C23:1, dl8:l-C24:l, and dl8:l-C24:0. Taken together with previous studies, these findings in human medulloblastoma cells support the view that high expression and marked heterogeneity of ceramide structure are general characteristics of tumor gangliosides, molecules which are shed by the tumor cells and which are biologically active in vivo.

Experimental Cell Research, 1997

Among the tumor -host interactions believed to fa-Shedding of immunosuppressive gangliosides is a... more Among the tumor -host interactions believed to fa-Shedding of immunosuppressive gangliosides is an vor tumor cell survival, one process is the release or important characteristic of both experimental and hu-shedding of tumor cell surface molecules which may man tumors. Using a medulloblastoma cell line, Daoy, have important biological properties . We are studywith a very high ganglioside expression (141 { 13 nmol/ ing a specific class of cell surface glycosphingolipids, 10 8 cells) and a well-characterized ganglioside complegangliosides, in this context. Gangliosides consist of a ment, we have now studied ganglioside shedding by sialic acid-containing carbohydrate portion and a lipid human brain tumor cells. Shedding of gangliosides, portion (ceramide) embedded in the cell membrane. quantified by metabolic radiolabeling, was significant Ganglioside molecules have a number of important bi-(169 pmol/10 8 cells/h) and was generalized with respect ological properties which could conceivably into the major ganglioside carbohydrate structures (G M2 , fluence the survival of the tumor cells which carry G M3 , and G D1a ). For each ganglioside, however, shedthese molecules. They have been shown to be immunoding was selective for ceramide structures containing suppressive [10-12], to enhance tumor formation in shorter fatty acyl chains. Rapid and ceramide-selecvivo , and to enhance platelet activation . Tutive shedding was confirmed in two additional human mor gangliosides have also been shown to have angiomedulloblastoma cell lines, D341 Med and D283 Med genic activity , which could promote neovascu-(112 and 59 pmol/10 8 cells/h). Significant ganglioside larization of a tumor bed. shedding is therefore a common characteristic of hu-Given the above biologically significant activities, the man medulloblastoma cells and may influence the bioultimate relevance of tumor gangliosides to tumor forlogical behavior of this tumor, in view of immunosupmation nevertheless depends on their access or availpressive and other biological properties of shed gangliability to ''sites of action.'' In other words, in order to osides. ᭧ 1997 Academic Press