Alla Danilkovitch - Academia.edu (original) (raw)

Papers by Alla Danilkovitch

Human mesenchymal stem cells (hMSCs) are rare progenitor cells present in adult bone marrow that ... more Human mesenchymal stem cells (hMSCs) are rare progenitor cells present in adult bone marrow that have the capacity to differentiate into a variety of tissue types, including bone, cartilage, tendon, fat, and muscle. In addition to multilineage differentiation capacity, MSCs regulate immune and inflammatory responses, providing therapeutic potential for treating diseases characterized by the presence of an inflammatory component. The availability of bone marrow and the ability to isolate and expand hMSCs ex vivo make these cells an attractive candidate for drug development. The low immunogenicity of these cells suggests that hMSCs can be transplanted universally without matching between donors and recipients. MSCs universality, along with the ability to manufacture and store these cells long-term, present a unique opportunity to produce an "off-the-shelf" cellular drug ready for treatment of diseases in acute settings. Accumulated animal and human data support MSC therapeutic potential for inflammatory diseases. Several phase III clinical trials for treatment of acute Graft Versus Host Disease (GVHD) and Crohn's disease are currently in progress. The current understanding of cellular and molecular targets underlying the mechanisms of MSCs action in inflammatory settings as well as clinical experience with hMSCs is summarized in this review.

Proceedings of The National Academy of Sciences, 2001

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple... more Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2͞LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for Ϸ25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.

Molecular and Cellular Biology, 2001

␤-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expressio... more ␤-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular ␤-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in ␤-catenin: mutations of ␤-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of ␤-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by ␤-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of ␤-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the ␤-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of ␤-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.

Genes Chromosomes & Cancer, 2000

The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other m... more The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other malignancies. It encodes a receptor tyrosine kinase (RTK) closely related to MET, whose mutations are associated with neoplasia. We investigated whether RON might be involved in the development or progression of lung cancer. We first determined the exon-intron structure of the gene by direct sequencing of RON cosmid DNA and PCR products containing intronic sequences, and then developed primers suitable for mutation analysis by the single-strand conformation polymorphism (SSCP) method. Twenty coding exons were characterized, all but the first one small (average size: 170 bp), a feature shared with other RTK genes. We performed SSCP analysis of RON in small and non-small cell lung cancer samples, upon detection of its expression in a sample of lung cancer cell lines. A mutation (T915C: L296P) was found in an adenocarcinoma specimen. Several single nucleotide polymorphisms were also found. The panel of intron-anchored primers developed in this work will be useful for mutation analysis of the RON gene in different types of human tumors.

Journal of Clinical Investigation, 2002

Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical for normal hemato... more Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical for normal hematopoiesis. Substitution of valine for aspartic acid 816 (D816V) constitutively actives human c-Kit, and this mutation is found in patients with mastocytosis, leukemia, and germ cell tumors. Immortalized murine progenitor cells (MIHCs) transduced with wild-type c-Kit proliferate in response to SCF, whereas cells expressing D816V c-Kit (MIHC-D816V) are factor-independent and tumorigenic. However, the mechanisms mediating transformation by D816V c-Kit are unknown. The objective of this study was to identify signaling components that contribute to D816V c-Kit-mediated transformation. SCF stimulates association of p85 PI3K with phosphorylated tyrosine 721 of wild-type c-Kit. Phosphatidylinositol 3 kinase (PI3K) subsequently contributes to the activation of Akt and Jnks. In contrast, these studies demonstrated that the D816V c-Kit mutant was constitutively associated with phosphorylated p85 PI3K , and, downstream of PI3K, Jnk 1 and Jnk 2 were activated but Akt was not. Interestingly, Erks 1 and 2 were not constitutively activated by D816V c-Kit. Thus, D816V c-Kit maintains the activity of PI3K but not of all signaling pathways activated by wild-type c-Kit. Further, all pathways downstream of PI3K are not constitutively active in MIHC-D816V cells. Studies with a PI3K inhibitor and D816V/Y721F c-Kit, a mutant incapable of recruiting PI3K, indicate that constitutive activation of PI3K through direct recruitment by D816V c-Kit plays a role in factorindependent growth of MIHC and is critical for tumorigenicity. (Blood. 2001;98: 1365-1373)

Journal of Immunological Methods, 2004

Herceptin, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, ... more Herceptin, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, is used for treatment of metastatic breast cancer patients overexpressing HER2 on tumor cells, and is being studied in clinical trials for therapy of other types of cancer. In the present work, we developed a Herceptin enzyme-linked immunosorbent assay (ELISA) from commercially available reagents to meet the growing needs of clinical studies. In this immunoassay, a mixture of monoclonal antibodies specific for the cytoplasmic domain of human HER2 (676-1255 amino acids) is adsorbed onto a microtiter plate, followed by addition of full-length HER2 protein, which is captured by the antibodies. Herceptin in the serum that is added to the microwells binds to the extracellular domain (ECD) of the captured HER2. The Herceptin bound to HER2 is detected by an antihuman IgG-horse radish peroxidase (HRP) conjugate and a (3, 5, 3V, 5V)-tetramethylenbenzidine (TMB) substrate. The calibration range of the assay is 5-100 ng/mL after 1:2000 sample dilution corresponding to 10-200 Ag/mL Herceptin in undiluted serum. The intra-and interassay CVs ranged from 4.56% to 13.3% and from 9.9% to 18.9%, respectively. The assay shows dilutional linearity and specificity. Soluble p105 HER2, which can be shed into serum does not interfere with the assay. The analytical performance of the Herceptin ELISA indicates that this assay can be used for monitoring concentration levels of Herceptin in human serum. D

Oncogene, 2007

The gene for tyrosine-kinase receptor Ron (MST1R) resides in the chromosome 3p21.3 region, freque... more The gene for tyrosine-kinase receptor Ron (MST1R) resides in the chromosome 3p21.3 region, frequently affected in common human malignancies. The gene generates two transcripts, 5 and 2 kb-long, full-length Ron (flRon) and short-form Ron (sfRon), respectively. Here, we show for the first time that the variegated Ron expression is associated with variations in the methylation patterns of two distinct CpG islands in Ron proximal promoter. Widespread hypermethylation associates with lack of flRon whereas hypermethylation of the distal island associates with transcription of sfRon, a constitutively active tyrosine-kinase that drives cell proliferation. sfRon inhibition with kinase-dead transgenes decreases cancer cell growth and induces cellular differentiation. sfRon could be a new drug target in cancer types in which it contributes to tumor progression.

Proceedings of The National Academy of Sciences, 2003

The candidate tumor-suppressor gene hyaluronidase 2 (HYAL2) encodes a glycosylphosphatidylinosito... more The candidate tumor-suppressor gene hyaluronidase 2 (HYAL2) encodes a glycosylphosphatidylinositol-anchored cell-surface protein that serves as an entry receptor for jaagsiekte sheep retrovirus, a virus that causes contagious lung cancer in sheep that is morphologically similar to human bronchioloalveolar carcinoma. The viral envelope (Env) protein alone can transform cultured cells, and we hypothesized that Env could bind and sequester the HYAL2 receptor and thus liberate a potential oncogenic factor bound and negatively controlled by HYAL2. Here we show that the HYAL2 receptor protein is associated with the RON receptor tyrosine kinase (also called MST1R or Stk in the mouse), rendering it functionally silent. In human cells expressing a jaagsiekte sheep retrovirus Env transgene, the Env protein physically associates with HYAL2. RON liberated from the association with HYAL2 becomes functionally active and consequently activates the Akt and mitogen-activated protein kinase pathways leading to oncogenic transformation of immortalized human bronchial epithelial cells. We find activated RON in a subset of human bronchioloalveolar carcinoma tumors, suggesting RON involvement in this type of human lung cancer.

Current Cancer Drug Targets, 2003

RON (Receptuer d'Origine Nantaise) is a member of the MET receptor tyrosine kinas... more RON (Receptuer d'Origine Nantaise) is a member of the MET receptor tyrosine kinase family. RON is expressed in various cell types including macrophages, epithelial and hematopoietic cells. Its ligand, macrophage stimulating protein (MSP, also known as hepatocyte growth factor-like protein), is a multifunctional factor regulating cell growth and survival, adhesion and motility, cytokine production and phagocytosis. Accumulated data indicate that in addition to the regulation of normal cell functions, RON can be involved in cancer development and progression: (i). RON is overexpressed and constitutively active in some primary tumors and tumor cell lines; (ii). experimental mutations of RON cause oncogenic cell transformation, and (iii). RON mediates susceptibility to Friend-virus-induced erythroleukemia in mice. Constitutive activation of intracellular signaling pathways such as the PI-3 kinase/AKT, beta-catenin, MAPK and JNK pathways may underlie the molecular mechanism of RON-mediated oncogenic cell transformation. The present review describes RON-activated signaling pathways, which may play an important role in tumor formation and metastasis.

Experimental Hematology, 2000

The apoptosis induced by actinomycin D (AD) in human megakaryoblastic leukemia CMK-7 cell line is... more The apoptosis induced by actinomycin D (AD) in human megakaryoblastic leukemia CMK-7 cell line is accelerated by microtubule poisons such as colcemid. We previously reported that the cell fragmentation in this apoptosis was suppressed by cytochalasin D (CD). 1 Here, we report that the AD-induced apoptosis is also accelerated by CD. The caspase-3 activation and DNA cleavage reached the maximum at least 10 hr earlier in the presence of CD than without this actin polymerization inhibitor. Decrease in organelle membrane potential, cytochrome c release from mitochondria, and cleavage of procaspase-9 to its active form began to appear prior to the caspase-3 activation. Apaf-1 was detected in the cytosol. These results suggest that the autocatalytic activation of caspase-9 starts the usual caspase cascade, and that the determinant, cytochrome c liberation, is promoted by interfering with the actin system. The cell surface F-actin disappeared by the AD-CD treatment and instead broad actin fibers appeared in the nucleus. These fibers were remote from the condensed chromatins, while F-actin in the cells undergoing apoptosis without CD accumulated around the nuclear fragments. The acceleration of AD-induced apoptosis by CD was found in U937 cells as well. Apoptosis induced by other DNA damaging agents (CDDP and MMC) was also accelerated by CD. The apoptosis by AD and CD was counteracted by antioxidants such as ␣ -tocopherol and luteolin. Thus, the DNA damage-induced apoptosis is affected by disruption of the actin system, and probably, reactive oxygen species are involved in the process.

Apoptosis, 2001

MSP is a serum protein belonging to the plasminogen-related kringle domain protein family. In add... more MSP is a serum protein belonging to the plasminogen-related kringle domain protein family. In addition to macrophages, epithelial cells are also MSP targets. MSP is a multifunctional factor regulating cell adhesion and motility, growth and survival. MSP mediates its biological activities by activating a transmembrane receptor tyrosine kinase called RON in humans or SKT in mice. MSP can protect epithelial cells from apoptosis by activating two independent signals in the PI3-K/AKT or the MAPK pathway. The MAPK pathway mediates the MSP anti-apoptotic effect only if additional signaling pathways are activated through adhesion. This indicates that MSP receptors and integrins, the receptors mediating cell-matrix-dependent adhesion, can collaborate in promotion of cell survival. This adhesion-dependent pathway, which is essential for the MAPK-mediated anti-apoptotic effect, remains to be identified. A hypothesis that Stat3 might represent a key component of the adhesion-induced anti-apoptotic pathway is presented in this review.

American Journal of Pathology, 2001

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Li... more An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Journal of Clinical Investigation, 2002

Hybridoma, 1997

We have studied an influence of nine overlapping peptides from the region between amino acid resi... more We have studied an influence of nine overlapping peptides from the region between amino acid residues 124 and 144 of the human interferon (FIN)-alpha 2 molecule on the growth of human lymphoid tumor cell lines in vitro. It was found that several, but not all, synthetic peptides inhibited proliferation of the same cell lines that IFN did. One of peptides, corresponding to the 124-138 amino acid residues of the IFN molecule (124-138) was most active. Using a human-mouse somatic hybrid cell line, we have shown that antiproliferative activity of the peptide 124-138 and IFN depended on the presence of human chromosome 21. Receptor binding studies also demonstrated that the peptide specifically interacted with membrane receptors on hybrid human-mouse cells carrying human chromosome 21, but not on parental mouse cells. Displacement experiments confirm that IFN and the peptide 124-138 compete for the same binding sites. Taken together, the data presented support a hypothesis that the C-terminal part of the IFN molecule contributes to antiproliferative activity possessed by IFN. Synthetic peptides studied in the present work may serve as a tool for studying tumor cell growth regulation by IFN and may be considered as potential nontoxic anti-tumor agents.

Molecular and Cellular Biology, 2000

In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a grow... more In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a growth and motility factor for epithelial cells. The growth and survival of epithelial cells generally require two signals, one generated by interaction with extracellular matrix via integrins, the other initiated by a growth factor. Therefore we investigated the effect of MSP on epithelial cell survival. Survival of epithelial cells cultured overnight in serum-free medium was promoted by adhesion, which activated both the phosphatidylinositol 3-kinase (PI3-K)/AKT and mitogen-activated protein kinase (MAPK) pathways, operating independently of one another. The number of apoptotic cells resulting from inhibition of either pathway alone was approximately doubled by simultaneous inhibition of both pathways. This shows that each pathway made a partial contribution to the prevention of apoptosis. In the presence of an inhibitor of either pathway, MSP increased the activity of the other pathway so that the single uninhibited pathway alone was sufficient to prevent apoptosis.

Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are pla... more Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors. Each one is a heterodimeric protein comprising a disulfide-linked ␣ chain and a serine protease-like ␤ chain. Despite structural similarities between MSP and HGF, the primary receptor binding site is located on the ␣ chain of HGF/SF but on the ␤ chain of MSP. To obtain insight into the structural basis for MSP ␤ chain binding, ␤ chain structure was modeled from coordinates of an existing model of the HGF ␤ chain. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn 682 /Glu 648 interacting pair, leaving a shallow cavity for possible ␤ chain interaction with the receptor. Mutants in this region were created, and their binding characteristics were determined. A double mutation of Asn 682 /Glu 648 caused diminished binding of the ␤ chain to the MSP receptor, and a single mutation of neighboring Arg 683 completely abolished binding. Thus, this region of the molecule is critical for binding. We also found that at equimolar concentrations of free ␣ and ␤ chains, ␣ chain binding to receptor was detectable, at levels considerably lower than ␤ chain binding. The EC 50 values determined by quantitative enzyme-linked immunosorbent assay are 0.25 and 16.9 nM for ␤ and ␣ chain, respectively. The data suggest that MSP has two independent binding sites with high and low affinities located in ␤ and ␣ chain, respectively, and that the two sites together mediate receptor dimerization and subsequent activation.

Advances in Cancer Research, 1999

Macrophage stimulating protein (MSP) belongs to the plasminogen-related kringle domain family. In... more Macrophage stimulating protein (MSP) belongs to the plasminogen-related kringle domain family. In addition to stimulation of macrophages, MSP acts on other cell types including epithelial and hematopoietic cells. The MSP receptor is a transmembrane tyrosine kinase called RON in humans and STK in mice. MSP/receptor interaction induces activation of signal transduction pathways that mediate MSP biological activities. Cytoplasmic kinases are intracellular messengers occupying an important role in signal transduction. We have identified kinases that participate in RON signaling. In addition to previously identified involvement of phosphatidylinositol 3-kinase (PI3-K), JNK, and MAPK, we found that FAK, c-Src, and AKT are rapidly and transiently activated by MSP. FAK, MAPK, and c-Src are involved in MSP-induced cell proliferation. MAPK and c-Src are components of one signal transduction cascade, and MAPK is downstream of c-Src. FAK also regulates MSPinduced cell growth, but via a path different from c-Src/MAPK. AKT kinase is a component of a separate branch of the RON/PI3-K pathway that mediates the MSP anti-apoptotic effect on epithelial cells. PI3-K regulates MSP-induced adhesion and motility but via downstream components different from AKT. Thus, occupancy of the RON receptor by MSP activates distinct signal transduction pathways that mediate several cellular responses. J. Leukoc. Biol. 65: 345-348; 1999.

Experimental Cell Research, 1999

Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity v... more Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity via the RON/STK receptor tyrosine kinase. MSP promotes integrin-dependent epithelial cell migration, which suggests that MSP may regulate integrin receptor functions. Integrins are cell surface receptors for extracellular matrix. Epithelial cell adhesion and motility are mediated by integrins. We studied the enhancement by MSP of cell adhesion and the molecular mechanisms mediating this effect. MSP decreased the time required for adhesion of 293 and RE7 epithelial cells to substrates coated with collagen or fibronectin.

Human mesenchymal stem cells (hMSCs) are rare progenitor cells present in adult bone marrow that ... more Human mesenchymal stem cells (hMSCs) are rare progenitor cells present in adult bone marrow that have the capacity to differentiate into a variety of tissue types, including bone, cartilage, tendon, fat, and muscle. In addition to multilineage differentiation capacity, MSCs regulate immune and inflammatory responses, providing therapeutic potential for treating diseases characterized by the presence of an inflammatory component. The availability of bone marrow and the ability to isolate and expand hMSCs ex vivo make these cells an attractive candidate for drug development. The low immunogenicity of these cells suggests that hMSCs can be transplanted universally without matching between donors and recipients. MSCs universality, along with the ability to manufacture and store these cells long-term, present a unique opportunity to produce an "off-the-shelf" cellular drug ready for treatment of diseases in acute settings. Accumulated animal and human data support MSC therapeutic potential for inflammatory diseases. Several phase III clinical trials for treatment of acute Graft Versus Host Disease (GVHD) and Crohn's disease are currently in progress. The current understanding of cellular and molecular targets underlying the mechanisms of MSCs action in inflammatory settings as well as clinical experience with hMSCs is summarized in this review.

Proceedings of The National Academy of Sciences, 2001

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple... more Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2͞LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for Ϸ25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.

Molecular and Cellular Biology, 2001

␤-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expressio... more ␤-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular ␤-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in ␤-catenin: mutations of ␤-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of ␤-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by ␤-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of ␤-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the ␤-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of ␤-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.

Genes Chromosomes & Cancer, 2000

The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other m... more The human RON gene (MST1R) maps to 3p21.3, a region frequently altered in lung cancer and other malignancies. It encodes a receptor tyrosine kinase (RTK) closely related to MET, whose mutations are associated with neoplasia. We investigated whether RON might be involved in the development or progression of lung cancer. We first determined the exon-intron structure of the gene by direct sequencing of RON cosmid DNA and PCR products containing intronic sequences, and then developed primers suitable for mutation analysis by the single-strand conformation polymorphism (SSCP) method. Twenty coding exons were characterized, all but the first one small (average size: 170 bp), a feature shared with other RTK genes. We performed SSCP analysis of RON in small and non-small cell lung cancer samples, upon detection of its expression in a sample of lung cancer cell lines. A mutation (T915C: L296P) was found in an adenocarcinoma specimen. Several single nucleotide polymorphisms were also found. The panel of intron-anchored primers developed in this work will be useful for mutation analysis of the RON gene in different types of human tumors.

Journal of Clinical Investigation, 2002

Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical for normal hemato... more Stem cell factor (SCF) binds the receptor tyrosine kinase c-Kit and is critical for normal hematopoiesis. Substitution of valine for aspartic acid 816 (D816V) constitutively actives human c-Kit, and this mutation is found in patients with mastocytosis, leukemia, and germ cell tumors. Immortalized murine progenitor cells (MIHCs) transduced with wild-type c-Kit proliferate in response to SCF, whereas cells expressing D816V c-Kit (MIHC-D816V) are factor-independent and tumorigenic. However, the mechanisms mediating transformation by D816V c-Kit are unknown. The objective of this study was to identify signaling components that contribute to D816V c-Kit-mediated transformation. SCF stimulates association of p85 PI3K with phosphorylated tyrosine 721 of wild-type c-Kit. Phosphatidylinositol 3 kinase (PI3K) subsequently contributes to the activation of Akt and Jnks. In contrast, these studies demonstrated that the D816V c-Kit mutant was constitutively associated with phosphorylated p85 PI3K , and, downstream of PI3K, Jnk 1 and Jnk 2 were activated but Akt was not. Interestingly, Erks 1 and 2 were not constitutively activated by D816V c-Kit. Thus, D816V c-Kit maintains the activity of PI3K but not of all signaling pathways activated by wild-type c-Kit. Further, all pathways downstream of PI3K are not constitutively active in MIHC-D816V cells. Studies with a PI3K inhibitor and D816V/Y721F c-Kit, a mutant incapable of recruiting PI3K, indicate that constitutive activation of PI3K through direct recruitment by D816V c-Kit plays a role in factorindependent growth of MIHC and is critical for tumorigenicity. (Blood. 2001;98: 1365-1373)

Journal of Immunological Methods, 2004

Herceptin, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, ... more Herceptin, a humanized anti-human epidermal growth factor receptor-2 (HER2) monoclonal antibody, is used for treatment of metastatic breast cancer patients overexpressing HER2 on tumor cells, and is being studied in clinical trials for therapy of other types of cancer. In the present work, we developed a Herceptin enzyme-linked immunosorbent assay (ELISA) from commercially available reagents to meet the growing needs of clinical studies. In this immunoassay, a mixture of monoclonal antibodies specific for the cytoplasmic domain of human HER2 (676-1255 amino acids) is adsorbed onto a microtiter plate, followed by addition of full-length HER2 protein, which is captured by the antibodies. Herceptin in the serum that is added to the microwells binds to the extracellular domain (ECD) of the captured HER2. The Herceptin bound to HER2 is detected by an antihuman IgG-horse radish peroxidase (HRP) conjugate and a (3, 5, 3V, 5V)-tetramethylenbenzidine (TMB) substrate. The calibration range of the assay is 5-100 ng/mL after 1:2000 sample dilution corresponding to 10-200 Ag/mL Herceptin in undiluted serum. The intra-and interassay CVs ranged from 4.56% to 13.3% and from 9.9% to 18.9%, respectively. The assay shows dilutional linearity and specificity. Soluble p105 HER2, which can be shed into serum does not interfere with the assay. The analytical performance of the Herceptin ELISA indicates that this assay can be used for monitoring concentration levels of Herceptin in human serum. D

Oncogene, 2007

The gene for tyrosine-kinase receptor Ron (MST1R) resides in the chromosome 3p21.3 region, freque... more The gene for tyrosine-kinase receptor Ron (MST1R) resides in the chromosome 3p21.3 region, frequently affected in common human malignancies. The gene generates two transcripts, 5 and 2 kb-long, full-length Ron (flRon) and short-form Ron (sfRon), respectively. Here, we show for the first time that the variegated Ron expression is associated with variations in the methylation patterns of two distinct CpG islands in Ron proximal promoter. Widespread hypermethylation associates with lack of flRon whereas hypermethylation of the distal island associates with transcription of sfRon, a constitutively active tyrosine-kinase that drives cell proliferation. sfRon inhibition with kinase-dead transgenes decreases cancer cell growth and induces cellular differentiation. sfRon could be a new drug target in cancer types in which it contributes to tumor progression.

Proceedings of The National Academy of Sciences, 2003

The candidate tumor-suppressor gene hyaluronidase 2 (HYAL2) encodes a glycosylphosphatidylinosito... more The candidate tumor-suppressor gene hyaluronidase 2 (HYAL2) encodes a glycosylphosphatidylinositol-anchored cell-surface protein that serves as an entry receptor for jaagsiekte sheep retrovirus, a virus that causes contagious lung cancer in sheep that is morphologically similar to human bronchioloalveolar carcinoma. The viral envelope (Env) protein alone can transform cultured cells, and we hypothesized that Env could bind and sequester the HYAL2 receptor and thus liberate a potential oncogenic factor bound and negatively controlled by HYAL2. Here we show that the HYAL2 receptor protein is associated with the RON receptor tyrosine kinase (also called MST1R or Stk in the mouse), rendering it functionally silent. In human cells expressing a jaagsiekte sheep retrovirus Env transgene, the Env protein physically associates with HYAL2. RON liberated from the association with HYAL2 becomes functionally active and consequently activates the Akt and mitogen-activated protein kinase pathways leading to oncogenic transformation of immortalized human bronchial epithelial cells. We find activated RON in a subset of human bronchioloalveolar carcinoma tumors, suggesting RON involvement in this type of human lung cancer.

Current Cancer Drug Targets, 2003

RON (Receptuer d'Origine Nantaise) is a member of the MET receptor tyrosine kinas... more RON (Receptuer d'Origine Nantaise) is a member of the MET receptor tyrosine kinase family. RON is expressed in various cell types including macrophages, epithelial and hematopoietic cells. Its ligand, macrophage stimulating protein (MSP, also known as hepatocyte growth factor-like protein), is a multifunctional factor regulating cell growth and survival, adhesion and motility, cytokine production and phagocytosis. Accumulated data indicate that in addition to the regulation of normal cell functions, RON can be involved in cancer development and progression: (i). RON is overexpressed and constitutively active in some primary tumors and tumor cell lines; (ii). experimental mutations of RON cause oncogenic cell transformation, and (iii). RON mediates susceptibility to Friend-virus-induced erythroleukemia in mice. Constitutive activation of intracellular signaling pathways such as the PI-3 kinase/AKT, beta-catenin, MAPK and JNK pathways may underlie the molecular mechanism of RON-mediated oncogenic cell transformation. The present review describes RON-activated signaling pathways, which may play an important role in tumor formation and metastasis.

Experimental Hematology, 2000

The apoptosis induced by actinomycin D (AD) in human megakaryoblastic leukemia CMK-7 cell line is... more The apoptosis induced by actinomycin D (AD) in human megakaryoblastic leukemia CMK-7 cell line is accelerated by microtubule poisons such as colcemid. We previously reported that the cell fragmentation in this apoptosis was suppressed by cytochalasin D (CD). 1 Here, we report that the AD-induced apoptosis is also accelerated by CD. The caspase-3 activation and DNA cleavage reached the maximum at least 10 hr earlier in the presence of CD than without this actin polymerization inhibitor. Decrease in organelle membrane potential, cytochrome c release from mitochondria, and cleavage of procaspase-9 to its active form began to appear prior to the caspase-3 activation. Apaf-1 was detected in the cytosol. These results suggest that the autocatalytic activation of caspase-9 starts the usual caspase cascade, and that the determinant, cytochrome c liberation, is promoted by interfering with the actin system. The cell surface F-actin disappeared by the AD-CD treatment and instead broad actin fibers appeared in the nucleus. These fibers were remote from the condensed chromatins, while F-actin in the cells undergoing apoptosis without CD accumulated around the nuclear fragments. The acceleration of AD-induced apoptosis by CD was found in U937 cells as well. Apoptosis induced by other DNA damaging agents (CDDP and MMC) was also accelerated by CD. The apoptosis by AD and CD was counteracted by antioxidants such as ␣ -tocopherol and luteolin. Thus, the DNA damage-induced apoptosis is affected by disruption of the actin system, and probably, reactive oxygen species are involved in the process.

Apoptosis, 2001

MSP is a serum protein belonging to the plasminogen-related kringle domain protein family. In add... more MSP is a serum protein belonging to the plasminogen-related kringle domain protein family. In addition to macrophages, epithelial cells are also MSP targets. MSP is a multifunctional factor regulating cell adhesion and motility, growth and survival. MSP mediates its biological activities by activating a transmembrane receptor tyrosine kinase called RON in humans or SKT in mice. MSP can protect epithelial cells from apoptosis by activating two independent signals in the PI3-K/AKT or the MAPK pathway. The MAPK pathway mediates the MSP anti-apoptotic effect only if additional signaling pathways are activated through adhesion. This indicates that MSP receptors and integrins, the receptors mediating cell-matrix-dependent adhesion, can collaborate in promotion of cell survival. This adhesion-dependent pathway, which is essential for the MAPK-mediated anti-apoptotic effect, remains to be identified. A hypothesis that Stat3 might represent a key component of the adhesion-induced anti-apoptotic pathway is presented in this review.

American Journal of Pathology, 2001

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Li... more An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Journal of Clinical Investigation, 2002

Hybridoma, 1997

We have studied an influence of nine overlapping peptides from the region between amino acid resi... more We have studied an influence of nine overlapping peptides from the region between amino acid residues 124 and 144 of the human interferon (FIN)-alpha 2 molecule on the growth of human lymphoid tumor cell lines in vitro. It was found that several, but not all, synthetic peptides inhibited proliferation of the same cell lines that IFN did. One of peptides, corresponding to the 124-138 amino acid residues of the IFN molecule (124-138) was most active. Using a human-mouse somatic hybrid cell line, we have shown that antiproliferative activity of the peptide 124-138 and IFN depended on the presence of human chromosome 21. Receptor binding studies also demonstrated that the peptide specifically interacted with membrane receptors on hybrid human-mouse cells carrying human chromosome 21, but not on parental mouse cells. Displacement experiments confirm that IFN and the peptide 124-138 compete for the same binding sites. Taken together, the data presented support a hypothesis that the C-terminal part of the IFN molecule contributes to antiproliferative activity possessed by IFN. Synthetic peptides studied in the present work may serve as a tool for studying tumor cell growth regulation by IFN and may be considered as potential nontoxic anti-tumor agents.

Molecular and Cellular Biology, 2000

In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a grow... more In addition to its effects on macrophage function, macrophage-stimulating protein (MSP) is a growth and motility factor for epithelial cells. The growth and survival of epithelial cells generally require two signals, one generated by interaction with extracellular matrix via integrins, the other initiated by a growth factor. Therefore we investigated the effect of MSP on epithelial cell survival. Survival of epithelial cells cultured overnight in serum-free medium was promoted by adhesion, which activated both the phosphatidylinositol 3-kinase (PI3-K)/AKT and mitogen-activated protein kinase (MAPK) pathways, operating independently of one another. The number of apoptotic cells resulting from inhibition of either pathway alone was approximately doubled by simultaneous inhibition of both pathways. This shows that each pathway made a partial contribution to the prevention of apoptosis. In the presence of an inhibitor of either pathway, MSP increased the activity of the other pathway so that the single uninhibited pathway alone was sufficient to prevent apoptosis.

Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are pla... more Macrophage-stimulating protein (MSP) and hepatocyte growth factor/scatter factor (HGF/SF) are plasminogen-related growth and motility factors that interact with cell-surface protein tyrosine kinase receptors. Each one is a heterodimeric protein comprising a disulfide-linked ␣ chain and a serine protease-like ␤ chain. Despite structural similarities between MSP and HGF, the primary receptor binding site is located on the ␣ chain of HGF/SF but on the ␤ chain of MSP. To obtain insight into the structural basis for MSP ␤ chain binding, ␤ chain structure was modeled from coordinates of an existing model of the HGF ␤ chain. The model revealed that the region corresponding to the S1 specificity pocket in trypsin is filled by the Asn 682 /Glu 648 interacting pair, leaving a shallow cavity for possible ␤ chain interaction with the receptor. Mutants in this region were created, and their binding characteristics were determined. A double mutation of Asn 682 /Glu 648 caused diminished binding of the ␤ chain to the MSP receptor, and a single mutation of neighboring Arg 683 completely abolished binding. Thus, this region of the molecule is critical for binding. We also found that at equimolar concentrations of free ␣ and ␤ chains, ␣ chain binding to receptor was detectable, at levels considerably lower than ␤ chain binding. The EC 50 values determined by quantitative enzyme-linked immunosorbent assay are 0.25 and 16.9 nM for ␤ and ␣ chain, respectively. The data suggest that MSP has two independent binding sites with high and low affinities located in ␤ and ␣ chain, respectively, and that the two sites together mediate receptor dimerization and subsequent activation.

Advances in Cancer Research, 1999

Macrophage stimulating protein (MSP) belongs to the plasminogen-related kringle domain family. In... more Macrophage stimulating protein (MSP) belongs to the plasminogen-related kringle domain family. In addition to stimulation of macrophages, MSP acts on other cell types including epithelial and hematopoietic cells. The MSP receptor is a transmembrane tyrosine kinase called RON in humans and STK in mice. MSP/receptor interaction induces activation of signal transduction pathways that mediate MSP biological activities. Cytoplasmic kinases are intracellular messengers occupying an important role in signal transduction. We have identified kinases that participate in RON signaling. In addition to previously identified involvement of phosphatidylinositol 3-kinase (PI3-K), JNK, and MAPK, we found that FAK, c-Src, and AKT are rapidly and transiently activated by MSP. FAK, MAPK, and c-Src are involved in MSP-induced cell proliferation. MAPK and c-Src are components of one signal transduction cascade, and MAPK is downstream of c-Src. FAK also regulates MSPinduced cell growth, but via a path different from c-Src/MAPK. AKT kinase is a component of a separate branch of the RON/PI3-K pathway that mediates the MSP anti-apoptotic effect on epithelial cells. PI3-K regulates MSP-induced adhesion and motility but via downstream components different from AKT. Thus, occupancy of the RON receptor by MSP activates distinct signal transduction pathways that mediate several cellular responses. J. Leukoc. Biol. 65: 345-348; 1999.

Experimental Cell Research, 1999

Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity v... more Macrophage stimulating protein (MSP) is a growth and motility factor that mediates its activity via the RON/STK receptor tyrosine kinase. MSP promotes integrin-dependent epithelial cell migration, which suggests that MSP may regulate integrin receptor functions. Integrins are cell surface receptors for extracellular matrix. Epithelial cell adhesion and motility are mediated by integrins. We studied the enhancement by MSP of cell adhesion and the molecular mechanisms mediating this effect. MSP decreased the time required for adhesion of 293 and RE7 epithelial cells to substrates coated with collagen or fibronectin.