ARTICLE |
Correspondence to: Hiroshi Kitamura, Laboratory of Biochemistry, Dept. of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. E-mail: ktmr@vetmed.hokudai.ac.jp
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We isolated cDNA of the mouse homologue of the src-suppressed C kinase substrate (SSeCKS) and analyzed the effects of lipopolysaccharide (LPS) injection on the tissue expression pattern of this protein. Northern blotting analysis showed that SSeCKS mRNA was expressed abundantly in the testis but at undetectable levels in other tissues of untreated control mice. Intraperitoneal administration of LPS strongly induced SSeCKS mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, adrenal gland, and pituitary gland, as well as in the brain. In lung and spleen, the SSeCKS mRNA levels increased almost 10-fold at 1 hr after LPS injection and persisted at high levels until 4 hr. Both in situ hybridization and immunohistochemical studies revealed that LPS administration conspicuously elevated expression of SSeCKS mRNA and protein in vascular endothelial cells of several organs. Ectopic expression of SSeCKS caused loss of cytoplasmic F-actin fibers in the mouse endothelial cell line LEII. These results indicate that SSeCKS is one of the major LPS-responsive proteins and may participate in alteration of cytoskeletal architecture in endothelial cells during inflammation. (J Histochem Cytochem 50:245255, 2002)
Key Words: SSeCKS, lipopolysaccharide, endothelial cells, reticular cells, inflammation, protein kinase C, protein kinase A, MARCKS, gravin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LIPOPOLYSACCHARIDE (LPS), a component of the outer membrane of Gram-negative bacteria, is a potent inducer of systemic inflammation (
Src-suppressed C kinase substrate (SSeCKS) was originally isolated from rat cell lines by
We have searched for inflammation-responsive genes in the mouse brain by differential display analysis and identified 11 of about 1500 genes whose mRNA levels were elevated by IP administration of LPS (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental Animals and Treatments
Male C57BL/6 mice (78 weeks old; SLC, Shizuoka, Japan) and Wistar rats (910 weeks old; Hokkaido University, Sapporo, Japan) were housed in plastic cages at 24 ± 1C on a 12-hr lightdark cycle (lights on at 0700 hr1900 hr) and given free access to laboratory chow and water. Some of them were injected IP with 13 mg/kg of LPS (E. coli 055:B5; Difco, Detroit, MI) or sterile PBS. The experimental procedure and care of the animals were in accordance with the guidelines of the Animal Care and Use Committee of Hokkaido University.
Cloning of Mouse SSeCKS cDNA
A mouse testis TriplEX cDNA library (Clontech; Palo Alto, CA) was screened using [
32P]-dCTP-labeled cDNA of a primary isolated partial 131.5 fragment (corresponding to +4797 to +5551bp of mouse SSeCKS cDNA; accession number AB020886) and that of mouse SSeCKS (+146 to +875 bp) obtained by reverse transcription-polymerase chain reaction (RT-PCR) from mouse testis RNA using the following primers: 5'-CCAAGCTCCCACAGAAGAATG-3' and 5'-GCCCAACCGTGAGTGAAGAA-3' based on the reported sequences of rat SSeCKS (accession number U23146). Four positive clones were obtained and the remaining nucleotides were amplified by PCR using the
TriplEX cDNA library as a template. Sequences of fragments were determined using an ABI PRISM 377 genetic analyzer (Perkin Elmer Applied Biosystems; Foster City, CA).
Northern Blotting
Tissue samples from the mice and rats were taken before and at 0.512 hr after LPS or PBS injection. Total RNA was extracted with TRIzol solution (Gibco BRL; Gaithersburg, MD), denatured at 65C, separated on a 1% agaroseformaldehyde gel, and transferred to and fixed on a nylon membrane (Amersham Pharmacia; Piscataway, NJ). A cDNA fragment of rat SSeCKS (+2459 to +3159 bp) was prepared by RT-PCR from total RNA extracted from rat testis using the following primers: 5'-ATGAGGACGACCCTAATGTC-3' and 5'-CTCAACCTTCTCCAGTGCTT-3'. The cDNA probes of mouse SSeCKS (+4797 to +5551 bp) and rat SSeCKS (+2459 to +3159 bp) were labeled with [32P]-dCTP. Nylon membranes were prehybridized with a buffer containing 50% formamide, 5 x SSPE, 0.1% SDS, 5 x Denhardt's solution, and 200 µg/ml salmon sperm DNA at 42C for 10 hr, and then hybridized at 42C for 12 hr in the prehybridization buffer supplemented with 1.0 x 106 cpm/ml of the labeled probes. The blots were washed with 2 x SSC, 0.1% SDS, and 0.1 x SSC, 0.1% SDS solutions, and were exposed to X-ray films for 1 or 2 weeks. The radioactivity was quantified using a BAS-1000 bioimage analyzer (Fuji Film; Tokyo, Japan). The membranes were also hybridized with cDNA of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or mouse 18s ribosomal RNA (18s rRNA) as a reference.
In Situ Hybridization
Two non-overlapping sense and antisense oligonucleotides corresponding to mouse SSeCKS +4808 to +4852 bp and +4882 to +4926 bp were used as probes for in situ hybridization analysis. These oligonucleotides were labeled with [35S]-dATP using terminal deoxyribonucleotidyl transferase (Promega; Madison, WI). Tissues were freshly removed from mice at 2 hr after LPS or PBS injection and were frozen in liquid nitrogen. Cryostat sections 16 µm thick were prepared and mounted on glass slides pre-coated with 3-aminopropyltriethoxysilane. They were fixed with 4% paraformaldehyde for 10 min and acetylated for 10 min in 0.25% acetic anhydride in PBS. The sections were dehydrated through a graded series of ethanol and prehybridized for 1 hr in a buffer containing 50% formamide, 0.1 M Tris-HCl (pH 7.5), 4 x SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin (BSA), 0.6 M NaCl, 0.25% SDS, 200 µg/ml yeast transfer RNA, 1 mM EDTA, and 10% dextran sulfate. Hybridization was performed at 42C for 10 hr in the prehybridization buffer supplemented with 1.0 x 107 cpm/ml of the 35S-labeled oligonucleotide probes. The sections were washed at room temperature for 20 min in 2 x SSC containing 0.1% sarkosyl and at 55C for 40 min in 0.1 x SSC containing 0.1% sarkosyl. The sections were either exposed to Hypefilm-ßmax (Amersham Pharmacia Biotech) for 3 weeks or dipped in Kodak NTB2 nuclear track emulsion and exposed for 2 months. All sections were counterstained with hematoxylin.
Immunohistochemistry
Immunostaining of SSeCKS was carried out according to the avidinbiotin complex method with rabbit anti-rat SSeCKS serum (a kind gift from Dr. Irwin H. Gelman; Mount Sinai Medical Center, New York, NY). The crossreactivity of the serum against mouse SSeCKS was confirmed by
For immunoelectron microscopy, the liver, brain, spleen, and lung were processed for a pre-embedding silver-intensified immunogold method. Paraformaldehyde-fixed cryostat sections were incubated in the SSeCKS antiserum diluted 1:2000, and subsequently reacted with goat anti-rabbit IgG covalently linked to 1.4-nm gold particles (Nanogold; Nanoprobes, Stony Brook, NY). After silver enhancement (HQ silver; Nanoprobes), sections were osmificated, dehydrated, and embedded in Epon 812 (Nisshin EM; Tokyo, Japan). Ultrathin sections were prepared and stained with an aqueous solution of 2% uranyl acetate for observation under an electron microscope (JEM-100SX; JEOL, Tokyo, Japan).
Ectopic Expression of SSeCKS mRNA in Cultured Endothelial Cells
The SSeCKS mRNA and its antisense expression plasmids for transfection were constructed by subcloning the full-length mouse SSeCKS cDNA (+1 to +5055 bp) into pDEST12.2 (Gibco) using the Gateway system (Gibco) according to the manufacturer's directions. Mouse endothelial cell line LEII was kindly provided by Dr. A. S. G. Curtis (University of Glasgow, UK) and was cultured in Dulbecco's modified Eagle's medium (Sigma; St Louis, MO) containing 10% fetal calf serum. LEII cells were transfected with a construct for sense or antisense SSeCKS, or with control pDEST12.2 using FuGENE reagent (Roche; Branchburg, NJ) for 3 days. Some cells were subsequently incubated with G418 neomycin (500 µg/ml) for 2 weeks. After washing with PBS, cells were seeded onto glass coverslips and cultured overnight. The cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized in 0.25% Triton X-100 for 5 min. They were then incubated with the anti-SSeCKS serum (1:250) in 1% BSA-containing PBS for 2 hr, blocked with 3% BSA in PBS for 1 hr, and finally incubated with both fluorescein isothiocyanate-labeled goat anti-rabbit IgG (1:500; Wako, Osaka, Japan) and phalloidinrhodamine (1:80; Molecular Probes, Eugene, OR) for 1 hr in the dark. The coverslips were washed with PBS and observed with a confocal scanning fluorescence microscope (FLUOVIEW system; Olympus, Tokyo, Japan).
Statistics
All values were expressed as means ± SEM. Statistical comparison was done by analysis of variance, followed by Scheffe's F-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA Cloning of Mouse SSeCKS
We screened inflammation-responsive genes in the mouse brain by differential display analysis and found one cDNA clone, 131.5, that markedly increased after IP administration of LPS. The full-length 131.5 cDNA had an open reading frame of 5055 bp encoding a polypeptide of 1684 amino acids, which was 84.8% identical to rat SSeCKS (GenBank accession number U23146). The deduced polypeptides of 131.5 preserved four PKC phosphorylation motifs, four serine residues phosphorylated by PKC, one PKA-binding site, and one N-terminal myristylation signal as reported in rat SSeCKS (
|
SSeCKS mRNA Expression After LPS Administration
The mRNA expression of SSeCKS in mouse tissues was investigated by Northern blotting analysis (Fig 2). In control mice injected with PBS, SSeCKS mRNA was at undetectable or quite low levels in all tissues examined except testis. Testis showed abundant SSeCKS mRNA expression, which was not influenced by LPS injection. However, remarkable induction of SSeCKS mRNA was found in brain, lung, heart, liver, spleen, kidney, lymph node, adrenal gland, and pituitary gland at 2 hr after IP injection of LPS. Similar induction of SSeCKS mRNA by LPS injection was also observed in rats (Fig 2). Fig 3 shows the time-dependent expression of SSeCKS mRNA after LPS injection in mouse lung and spleen. The SSeCKS mRNA level rose 10-fold in lung and spleen at 14 hr and 12 hr after LPS injection, respectively, and declined to lower levels at 12 hr.
|
|
In Situ Hybridization of SSeCKS mRNA
Cellular localization of SSeCKS mRNA was examined in PBS- and LPS-injected mice by in situ hybridization using 35S-labeled antisense oligonucleotides as probes. Selective expression of SSeCKS mRNA was found only in testis in PBS-injected mice, whereas at 2 hr after LPS injection intense signals of SSeCKS mRNA appeared in many tissues, including pituitary gland, lymph node, adrenal gland, lung, kidney, heart, liver, lung, brain, and spleen (Fig 4). Two non-overlapping antisense oligonucleotide probes gave essentially the same labeling patterns, whereas no specific hybridization signals were obtained with sense oligonucleotide probes (Fig 4).
|
By light microscopy, SSeCKS mRNA in control mice was abundantly present at the luminal side of the seminiferous tubules in the testis (Fig 5a), although the expression level differed considerably among the individual tubules depending on the stage of spermatogenesis. LPS treatment did not affect the SSeCKS mRNA expression in the testis (Fig 5b). In the kidney in control mice, weak but significant signals of SSeCKS mRNA existed at the periphery of renal corpuscles, which corresponded to Bowman's capsules (Fig 5c). After LPS stimulation, signals in the kidney were not restricted to Bowman's capsules but appeared diffusely and intensely in the renal glomeruli (Fig 5d). Signals in other tissues did not exceed the background levels in the control, whereas they prominently increased at 2 hr after LPS injection (Fig 5e and Fig 5f). In the brain, the SSeCKS mRNA signal exhibited many linear structures in the parenchyma and along the brain surface, corresponding to vascular endothelial cells and pia mater, respectively (Fig 5f). The signals in the spleen and lymph node were recognized selectively in the red pulp and medullary sinuses, respectively (Fig 5g and Fig 5h). In the liver, intense signals of SSeCKS mRNA were localized in slender cells along the sinusoidal walls but not in hepatocytes (Fig 5i). The anterior pituitary showed a similar image: dispersed cells among endocrine cells were intensely labeled (Fig 5j). Diffuse signals were distributed throughout the lung, except for non-labeled bronchioles (Fig 5k), and in the adrenal cortex (Fig 5l) and heart (data not shown).
|
Immunohistochemical Localization of SSeCKS Protein
For detailed identification of SSeCKS-expressing cells, immunohistochemical examinations were conducted using a rabbit antiserum against rat SSeCKS protein. In the testis in control mice, the SSeCKS immunoreactivity existed only in elongated spermatids at the late stage of spermatogenesis, and this staining pattern was not changed by LPS injection (Fig 6a and Fig 6b). The immunoreactivity in the kidney was restricted to the epithelial cells lining Bowman's capsules in control mice, whereas it also appeared intensely in mesangial cells in LPS-injected mice (Fig 6c and Fig 6d). In other organs, the SSeCKS immunoreactivity was undetectable in control mice, in which it became noticeable after LPS injection, being strongest at 812 hr. The LPS-induced immunoreactivity was localized in endothelial cells and reticular cells. For example, at 8 hr after LPS injection intense immunoreactivity was observed in all types of vessels (arterioles, capillaries, and venules) in the brain (Fig 6e and Fig 6f) and in those of sinuses of the spleen (Fig 6g). LPS also induced SSeCKS immunoreactivity in reticular cells in the red pulp of the spleen and the medulla of the lymph node (Fig 6g and Fig 6h). The immunoreactive reticular cells in the lymph node were distributed in the lumina of the medullary sinuses and lined the sinus walls as lymphatic endothelium (Fig 6h). The endothelial cells of capillaries and sinusoidal capillaries were predominantly immunoreactive in the liver (Fig 6i), adenohypophysis (Fig 6j), lung (Fig 6k), heart (Fig 6l), and adrenal cortex (data not shown). Thus, the cellular localization of SSeCKS protein was almost identical to that of its mRNA revealed by in situ hybridization.
|
To confirm the localization of SSeCKS in the endothelial cells, we performed immunoelectron microscopy using the silver-intensified immunogold method. As shown in Fig 7, SSeCKSgold particles were detected mainly in the cytoplasmic region of sinusoidal endothelial cells in the liver. SSeCKS immunoreactivity was also confined to endothelial cells and was undetectable in pericytes, vascular smooth muscle cells, and parenchymal cells in the brain, spleen, and lung (data not shown).
|
F-actin Fibers in Endothelial Cells After Transfection with SSeCKS cDNA
It has been argued that SSeCKS regulates the cytoskeletal architecture in cultured fibroblasts and mesangial cells (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Using a differential display technique, we cloned mouse SSeCKS as one of the molecules whose expression was conspicuously induced in the brain and several visceral organs by IP administration of LPS. Similar to rat SSeCKS (
The most notable finding of the present study was that IP LPS injection induced expression of SSeCKS predominantly in vascular endothelial cells of several organs. The LPS-induced SSeCKS expression was demonstrated both at the mRNA level by Northern blotting and in situ hybridization analyses and at the protein level by immunohistochemical examination, i.e., LPS injection caused rapid (14 hr) and remarkable (10-fold) increases in mRNA expression, followed by the obvious appearance of immunoreactivity for SSeCKS in 812 hr. It should be emphasized that the SSeCKS expression in these cells was undetectable in the normal condition without LPS injection. Previously, Gelman and colleagues (
Cells expressing SSeCKS in response to LPS included reticular cells of the spleen and lymph node as well as endothelial cells of the adenohypophysis, lung, spleen, liver, and adrenal cortex. These cells are members of the reticuloendothelial system (RES) that take up exogenous substances such as lithium carmine, glycosaminoglycans, proteoglycans, collagen, lipoprotein, and denatured erythrocytes (
In the present study we failed to detect any increase of SSeCKS mRNA and protein in LEII cells, even after LPS stimulation. The paucity of SSeCKS induction by LPS may be attributable to unusual characteristics of this immortal and proliferative cell line. Alternatively, LPS itself might not induce SSeCKS, and endogenous humoral factors released from non-endothelial cells stimulated by LPS might participate in SSeCKS induction. One of the candidate factors is tumor necrosis factor (TNF), which is a vasoactive cytokine induced in macrophages in response to LPS (
Rodent SSeCKS shows a significant structural homology with human gravin (80% similar (
![]() |
Acknowledgments |
---|
Supported in part by grants-in-aid from the Ministry of Agriculture, Forestry and Fisheries (RCP-2000-4230) and from the Ministry of Education, Culture, Sports, Science and Technology of Japan (12760191).
We are grateful to Dr I. H. Gelman for his kind gift of the antiserum against rat SSeCKS. We are also grateful to Dr A. S. G. Curtis for providing LEII cells.
Received for publication April 3, 2001; accepted September 5, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allen LH, Aderem A (1995) A role for MARCKS, the alpha isozyme of protein kinase C and myosin I zymosan phagocytosis by macrophages. J Exp Med 182:829-840[Abstract]
Bannerman DD, Goldblum SE (1997) Endotoxin induces endothelial barrier dysfunction through protein tyrosine phosphorylation. Am J Physiol 273:L217-226
Camussi G, Turello E, Bussolino F, Baglioni C (1991) Tumor necrosis factor alters cytoskeletal organization and barrier function of endothelial cells. Int Arch Allergy Appl Immunol 96:84-91[Medline]
Chapline C, Cottom J, Tobin H, Hulmers J, Crabb J, Jaken S (1998) A major, transformation-sensitive PKC-binding protein is also a PKC substrate involved in cytoskeletal remodeling. J Biol Chem 273:19482-19489
Chapline C, Mousseau B, Ramsay K, Duddy S, Li Y, Kiley SC, Jaken S (1996) Identification of a major protein kinase C-binding protein and substrate in rat embryo fibroblasts. Decreased expression in transformed cells. J Biol Chem 271:6417-6422
Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt AM, Stern DM (1998) Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91:3527-3561
Defazio G, Nico B, Trojano M, Ribatti D, Giorelli M, Ricchiuti F, Martino D, Roncali L, Livrea P (2000) Inhibition of protein kinase C counteracts TNFalpha-induced intercellular adhesion molecule 1 expression and fluid phase endocytosis on brain microvascular endothelial cells. Brain Res 863:245-248[Medline]
Erlichman J, GutierrezJuarez R, Zucker S, Mei XH, Orr GA (1999) Developmental expression of the protein kinase C substrate/binding protein (clone 72/SSeCKS) in rat testis. Identification as a scaffolding protein containing an A-kinase-anchoring domain which is expressed during late-stage spermatogenesis. Eur J Biochem 263:797-805
Gelman IH, Lee K, Tombler E, Gordon R, Lin X (1998) Control of cytoskeletal architecture by the src-suppressed C kinase substrate, SSeCKS. Cell Motil Cytoskel 41:1-17[Medline]
Gelman IH, Tombler E, Vargas J, Jr. (2000) A role for SSeCKS, a major protein kinase C substrate with tumor suppressor activity, in cytoskeletal architecture, formation of migratory processes, and cell migration during embryogenesis. Histochem J 32:13-26[Medline]
Goldblum SE, Ding X, Campbell WJ (1993) TNF-alpha induces endothelial cell F-actin depolymerization, new actin synthesis, and barrier dysfunction. Am J Physiol 264:C894-905
Gordon T, Grove B, Loftus JC, O'Toole T, McMillan R, Lindstrom J, Ginsberg MH (1992) Molecular cloning and preliminary characterization of a novel cytoplasmic antigen recognized by myasthenia gravis sera. J Clin Invest 90:992-999[Medline]
Grove BD, Bowditch R, Del Zoppo G, Ginsberg MH (1994) Restricted endothelial cell expression of gravin in vivo. Anat Rec 239:231-242[Medline]
Kawai Y, Smedsrod B, Elvevold K, Wake K (1998) Uptake of lithium carmine by sinusoidal endothelial and Kupffer cells of the rat liver: new insights into the classical vital staining and the reticulo-endothelial system. Cell Tissue Res 292:395-410[Medline]
Kitamura H, Kanehira K, Okita K, Morimatsu M, Saito M (2000) MAIL, a novel nuclear IB protein that potentiates LPS-induced IL-6 production. FEBS Lett 485:53-56[Medline]
Lin X, Nelson PJ, Frankfort B, Tombler E, Johnson R, Gelman IH (1995) Isolation and characterization of a novel mitogenic regulatory gene, 322, which is transcriptionally suppressed in cells transformed by src and ras. Mol Cell Biol 15:2754-2762[Abstract]
Lin X, Tombler E, Nelson PJ, Ross M, Gelman IH (1996) A novel src- and ras-suppressed protein kinase c substrate associated with cytoskeletal architecture. J Biol Chem 271:28430-28438
Nauert JB, Klauck TM, Langeberg LK, Scott JD (1996) Gravin, an autoantigen recognized by serum from myasthenia gravis patients, is a kinase scaffold protein. Curr Biol 7:52-62
Nelson PJ, Gelman IH (1997) Cell-cycle regulated expression and serine phosphorylation of the myristylated protein kinase C substrate, SSeCKS: correlation with culture confluency, cell cycle phase and serum response. Mol Cell Biochem 175:233-241[Medline]
Nelson PJ, Moissoglu K, Vargas J, Jr, Klotman PE, Gelman IH (1999) Involvement of the protein kinase C substrate, SSeCKS, in the actin-based stellate morphology of mesangial cells. J Cell Sci 112:361-370
Praaningvan Dalen DP, de Leeuw AM, Brouwer A, Knook DL (1987) Rat liver endothelial cells have a greater capacity than Kupffer cells to endocytose N-acetylglucosamine- and mannose-terminated glycoproteins. Hepatology 7:672-679[Medline]
Seykora JT, Ravetch JV, Aderem A (1991) Cloning and molecular characterization of the murine macrophage "68-kDa" protein kinase C substrate and its regulation by bacterial lipopolysaccharide. Proc Natl Acad Sci USA 88:2505-2509[Abstract]
Smedsrod B, Pertoft H, Gustafson S, Laurent TC (1990) Scavenger functions of the liver endothelial cell. Biochem J 266:313-327[Medline]
Song JC, Hrnjez BJ, Farokhzad OC, Matthews JB (1999) PKC-epsilon regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS. Am J Physiol 277:C1239-1249
Terada M (1993) A novel role in the removal of blood-borne foreign bodies for pulmonary capillaries in the guinea pig. Virchows Arch 63:147-157. [B]
Thelen M, Rosen A, Nairn AC, Aderem A (1990) Tumor necrosis factor alpha modifies agonist-dependent responses in human neutrophils by inducing the synthesis and myristylation of a specific protein kinase C substrate. Proc Natl Acad Sci USA 87:5603-5607[Abstract]
Ulevitch RJ, Tobias PS (1995) Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 13:437-457[Medline]
Xia W, Unger P, Miller L, Nelson J, Gelman IH (2001) The Src-suppressed C kinase substrate, SSeCKS, is a potent metastatis inhibitor in prostate cancer. Cancer Res 61:5644-5651