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• W2000002361 abstract "We report the first correlation of Nm23 sequence and its tumor metastasis-suppressive capacity using site-directed mutagenesis and an in vitro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVBamneo), the vector containing the wild type nm23-H1, or the nm23-H1 vector encoding mutations at the following amino acids: serine 44, a phosphorylation site; proline 96, the k-pn mutation in the Drosophila nm23 homolog that causes developmental defects; histidine 118, involved in Nm23's nucleoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 transfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, serine 120 to glycine, and to a lesser extent serine 120 to alanine mutant nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abrogate the motility-suppressive phenotype of nm23-H1. No effect was observed on cellular proliferation, nor were the serine 44 to alanine nm23-H1 mutant transfectants motile, demonstrating the specificity of the data. The data identify the first structural motifs of nm23-H1 that influence its metastasis suppressive effect and suggest complex biochemical associations or activities in the Nm23 suppressive pathway. We report the first correlation of Nm23 sequence and its tumor metastasis-suppressive capacity using site-directed mutagenesis and an in vitro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVBamneo), the vector containing the wild type nm23-H1, or the nm23-H1 vector encoding mutations at the following amino acids: serine 44, a phosphorylation site; proline 96, the k-pn mutation in the Drosophila nm23 homolog that causes developmental defects; histidine 118, involved in Nm23's nucleoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 transfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, serine 120 to glycine, and to a lesser extent serine 120 to alanine mutant nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abrogate the motility-suppressive phenotype of nm23-H1. No effect was observed on cellular proliferation, nor were the serine 44 to alanine nm23-H1 mutant transfectants motile, demonstrating the specificity of the data. The data identify the first structural motifs of nm23-H1 that influence its metastasis suppressive effect and suggest complex biochemical associations or activities in the Nm23 suppressive pathway. INTRODUCTIONThe metastatic spread of tumor cells and complications arising from the treatment of established metastases are major contributors to cancer patient mortality. As a result of differential colony hybridization between related high and low metastatic potential murine K-1735 melanoma cell lines, we identified nm23 on the basis of reduced mRNA (1Steeg P.S. Bevilacqua G. Kopper L. Thorgeirsson U.P. Talmadge J.E. Liotta L.A. Sobel M.E. J. Natl. Cancer Inst. 1988; 80: 200-204Crossref PubMed Scopus (1293) Google Scholar) and protein (2Rosengard A.M. Krutzsch H.C. Shearn A. Biggs J.R. Barker E. Margulies I.M.K. King C.R. Liotta L.A. Steeg P.S. Nature. 1989; 342: 177-180Crossref PubMed Scopus (478) Google Scholar) levels in highly metastatic tumor cells. Three human nm23 genes have been identified, nm23-H1, nm23-H2, and nm23-DR (2Rosengard A.M. Krutzsch H.C. Shearn A. Biggs J.R. Barker E. Margulies I.M.K. King C.R. Liotta L.A. Steeg P.S. Nature. 1989; 342: 177-180Crossref PubMed Scopus (478) Google Scholar, 3Venturelli D. Martinez R. Melotti P. Casella I. Peschile C. Cucco C. Spampinato G. Darzynkiewicz Z. Calabretta B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7435-7439Crossref PubMed Scopus (156) Google Scholar, 4Stahl J.A. Leone A. Rosengard A.M. Porter L. King C.R. Steeg P.S. Cancer Res. 1991; 51: 445-449PubMed Google Scholar). Homologs of nm23 have been cloned in other species as follows: murine nm23-M1 and nm23-M2 (1Steeg P.S. Bevilacqua G. Kopper L. Thorgeirsson U.P. Talmadge J.E. Liotta L.A. Sobel M.E. J. Natl. Cancer Inst. 1988; 80: 200-204Crossref PubMed Scopus (1293) Google Scholar, 5Urano T. Takamiya K. Furukawa K. Shiku H. FEBS Lett. 1992; 309: 358-362Crossref PubMed Scopus (80) Google Scholar), rat NDPKb and NDPKa (6Kimura N. Shimada N. Nomura K. Watanabe K. J. Biol. Chem. 1990; 265: 15744-15749Abstract Full Text PDF PubMed Google Scholar, 7Shimada N. Ishikawa N. Munakata Y. Toda T. Watanabe K. Kimura N. J. Biol. Chem. 1993; 268: 2583-2589Abstract Full Text PDF PubMed Google Scholar), Drosophila awd (8Dearolf C. Hersperger E. Shearn A. Dev. Biol. 1988; 129: 159-168Crossref PubMed Scopus (117) Google Scholar, 9Dearolf C. Tripoulas N. Biggs J. Shearn A. Dev. Biol. 1988; 129: 169-178Crossref PubMed Scopus (96) Google Scholar), Myxococcus ndk (10Muñoz-Dorado J. Inouye M. Inouye S. J. Biol. Chem. 1990; 265: 2707-2712Abstract Full Text PDF PubMed Google Scholar), Escherichia coli ndk (11Hama H. Almaula N. Lerner C.G. Inouye S. Inouye M. Gene (Amst.). 1991; 105: 31-36Crossref PubMed Scopus (76) Google Scholar), and Dictyostelium gip17 and guk7.2 (12Troll H. Winckler T. Lascu I. Muller N. Saurin W. Veron M. Mutzel R. J. Biol. Chem. 1993; 268: 25469-25475Abstract Full Text PDF PubMed Google Scholar, 13Wallet V. Mutzel R. Troll H. Barzu O. Wurster B. Veron M. Lacombe M.L. J. Natl. Cancer Inst. 1990; 82: 1199-1202Crossref PubMed Scopus (223) Google Scholar).The correlation of reduced nm23 expression and high tumor metastatic potential has been confirmed in additional metastasis model systems (1Steeg P.S. Bevilacqua G. Kopper L. Thorgeirsson U.P. Talmadge J.E. Liotta L.A. Sobel M.E. J. Natl. Cancer Inst. 1988; 80: 200-204Crossref PubMed Scopus (1293) Google Scholar, 14Caligo M.A. Cipollini G. Valromita C.D. Bistocchi M. Bevilacqua G. Anticancer Res. 1992; 12: 969-973PubMed Google Scholar, 15Steeg P.S. Bevilacqua G. Pozzatti R. Liotta L.A. Sobel M.E. Cancer Res. 1988; 48: 6550-6554PubMed Google Scholar, 16Lakshmi M. Parker C. Sherbert G. Anticancer Res. 1993; 13: 299-304PubMed Google Scholar, 17Su Z. Austin V.N. Zimmer S.G. Fisher P.B. Oncogene. 1993; 8: 1211-1219PubMed Google Scholar) and in human tumor cohort studies of breast (18Bevilacqua G. Sobel M.E. Liotta L.A. Steeg P.S. Cancer Res. 1989; 49: 5185-5190PubMed Google Scholar, 19Hennessy C. Henry J. May F.E.B. Westly B. Angus B. Lennard T.W.J. J. Natl. Cancer Inst. 1991; 83: 281-285Crossref PubMed Scopus (318) Google Scholar, 20Barnes R. Masood S. Barker E. Rosengard A.M. Coggin D.L. Crowell T. King C.R. Porter-Jordan K. Wargotz E.S. Liotta L.A. Steeg P.S. Am. J. Pathol. 1991; 139: 245-250PubMed Google Scholar, 21Tokunaga Y. Urano T. Furukawa K. Kondo H. Kanematsu T. Shiku H. Int. J. Cancer. 1993; 55: 66-71Crossref PubMed Scopus (142) Google Scholar, 22Royds J.A. Stephenson T.J. Rees R.C. Shorthouse A.J. Silcocks P.B. J. Natl. Cancer Inst. 1993; 85: 727-731Crossref PubMed Scopus (119) Google Scholar, 23Cropp C. Lidereau R. Leone A. Liscia D. Cappa A. Campbell G. Barker E. Doussal V.L. Steeg P. Callahan R. J. Natl. Cancer Inst. 1994; 86: 1167-1169Crossref PubMed Scopus (33) Google Scholar, 24Hirayama R. Sawai S. Takagi Y. Mishima Y. Kimura N. Shimada N. Esaki Y. Kurashima C. Utsuyama M. Hirokawa K. J. Natl. Cancer Inst. 1991; 83: 1249-1250Crossref PubMed Scopus (150) Google Scholar), ovarian (25Kapitonovic S. Spaventi R. Vujsic S. Petrovic Z. Kurjak A. Pavelic Z. Gluckman J. Stambrook P. Pavelic K. Anticancer Res. 1995; 15: 587-590PubMed Google Scholar, 26Mandai M. Konishi I. Koshiyama M. Mori T. Arao S. Tashiro H. Okamura H. Nomura H. Hiai H. Fukumoto M. Cancer Res. 1994; 54: 1825-1830PubMed Google Scholar, 27Scambia G. Ferrandina G. Marone M. Panici P.B. Giannitelli C. Piantelli M. Leone A. Manusco S. J. Clin. Oncol. 1996; 14: 334-342Crossref PubMed Scopus (63) Google Scholar), cervical (28Ilijas M. Pavelic K. Sarcevic B. Kapitanovic S. Kurjak A. Stambrook P. Gluckman J. Pavelic Z. Int. J. Oncol. 1994; 5: 1455-1457PubMed Google Scholar, 29Mandai M. Konishi I. Koshiyama M. Komatsu T. Yamamoto S. Nanbu K. Mori T. Fukumoto M. Cancer. 1995; 75: 2523-2529Crossref PubMed Scopus (65) Google Scholar), gastric (30Kodera Y. Isobe K.-I. Yamauchi M. Kendoh K. Kimura N. Akiyama S. Itoh K. Nakashima I. Takagi H. Cancer. 1994; 73: 259-265Crossref PubMed Scopus (81) Google Scholar), and hepatocellular (31Boix L. Bruix J. Campo E. Sole M. Castells A. Fuster J. Rivera F. Cardesa A. Rodes J. Gasteroenterology. 1994; 107: 486-491Abstract Full Text PDF PubMed Scopus (31) Google Scholar, 32Nakayama T. Ohtsuru A. Nakao K. Shima M. Nakata K. Watanabe K. Ishii N. Kimura N. Nagataki S. J. Natl. Cancer Inst. 1992; 84: 1349-1354Crossref PubMed Scopus (164) Google Scholar, 33Yamaguchi A. Urano T. Goi T. Takeuchi K. Niimoto S. Nakagawara G. Furukawa K. Shiku H. Cancer. 1994; 73: 2280-2284Crossref PubMed Scopus (87) Google Scholar) carcinomas and melanomas (35Flørenes V.A. Aamdal S. Myklebost O. Maelandsmo M. Bruland Ø.S. Fodstad Ø. Cancer Res. 1992; 52: 6088-6091PubMed Google Scholar). 1J. Royds, E. Barrett, R. Rees, S. Cross, and T. Stephenson, submitted for publication. In other cell types nm23 expression levels have been unrelated to or directly correlated with tumor metastatic potential, involved in cell growth, or altered in other ways, such as by mutation (reviewed in 36De La Rosa A. Williams R.L. Steeg P.S. BioEssays. 1995; 17: 53-62Crossref PubMed Scopus (225) Google Scholar), suggesting multiple or complex biological functions of the gene product(s).The functional involvement of nm23 gene expression in tumor metastatic potential was determined by its transfection into melanoma and breast carcinoma cell lines, which resulted in a 44-96% reduction in metastatic potential in vivo (37Leone A. Flatow U. King C.R. Sandeen M.A. Margulies I.M.K. Liotta L.A. Steeg P.S. Cell. 1991; 65: 25-35Abstract Full Text PDF PubMed Scopus (544) Google Scholar, 38Leone A. Flatow U. VanHoutte K. Steeg P.S. Oncogene. 1993; 8: 2325-2333PubMed Google Scholar, 39Parhar R.S. Shi Y. Zou M. Farid N.R. Ernst P. Al-Sedairy S. Int. J. Cancer. 1995; 60: 204-210Crossref PubMed Scopus (97) Google Scholar, 40Baba H. Urano T. Okada K. Furukawa K. Nakayama E. Tanaka H. Iwasaki K. Shiku H. Cancer Res. 1995; 55: 1977-1981PubMed Google Scholar, 41Fukuda M. Ishii A. Yasutomo Y. Shimada N. Ishikawa N. Hanai N. Nagara N. Irimura T. Nicolson G. Kimura N. Int. J. Cancer. 1996; 65: 531-537Crossref PubMed Scopus (64) Google Scholar). In vitro characterization of control- and nm23-transfected cell lines indicated changes in motility in Boyden chamber assays (42Kantor J.D. McCormick B. Steeg P.S. Zetter B.R. Cancer Res. 1993; 53: 1971-1973PubMed Google Scholar), invasiveness through Matrigel-coated filters (39Parhar R.S. Shi Y. Zou M. Farid N.R. Ernst P. Al-Sedairy S. Int. J. Cancer. 1995; 60: 204-210Crossref PubMed Scopus (97) Google Scholar), immunosensitivity to lymphokine-activated killer cells (39Parhar R.S. Shi Y. Zou M. Farid N.R. Ernst P. Al-Sedairy S. Int. J. Cancer. 1995; 60: 204-210Crossref PubMed Scopus (97) Google Scholar), and the colonization response in soft agar to tumor growth factor-β (37Leone A. Flatow U. King C.R. Sandeen M.A. Margulies I.M.K. Liotta L.A. Steeg P.S. Cell. 1991; 65: 25-35Abstract Full Text PDF PubMed Scopus (544) Google Scholar, 38Leone A. Flatow U. VanHoutte K. Steeg P.S. Oncogene. 1993; 8: 2325-2333PubMed Google Scholar). Cell proliferation rates were uniformly unchanged (37Leone A. Flatow U. King C.R. Sandeen M.A. Margulies I.M.K. Liotta L.A. Steeg P.S. Cell. 1991; 65: 25-35Abstract Full Text PDF PubMed Scopus (544) Google Scholar, 38Leone A. Flatow U. VanHoutte K. Steeg P.S. Oncogene. 1993; 8: 2325-2333PubMed Google Scholar, 40Baba H. Urano T. Okada K. Furukawa K. Nakayama E. Tanaka H. Iwasaki K. Shiku H. Cancer Res. 1995; 55: 1977-1981PubMed Google Scholar), providing evidence for specificity in biological function.Studies with homologs of nm23 in other species have provided evidence for a role in development and differentiation. Mutation or reduced expression of Drosophila abnormal wing discs (awd) resulted in abnormal cell morphology, aberrant differentiation, and cell necrosis postmetamorphosis (8Dearolf C. Hersperger E. Shearn A. Dev. Biol. 1988; 129: 159-168Crossref PubMed Scopus (117) Google Scholar, 9Dearolf C. Tripoulas N. Biggs J. Shearn A. Dev. Biol. 1988; 129: 169-178Crossref PubMed Scopus (96) Google Scholar). In the human MDA-MB-435 breast carcinoma model system, cells transfected with nm23-H1 exhibited both morphological and biosynthetic aspects of mammary differentiation upon three-dimensional culture in a basement membrane extract (43Howlett A.R. Petersen O.W. Steeg P.S. Bissell M.J. J. Natl. Cancer Inst. 1994; 86: 1838-1844Crossref PubMed Scopus (139) Google Scholar).The biochemical mechanism(s) mediating nm23 modulation of tumor metastatic potential and differentiation are unknown. A plethora of biochemical activities have been confirmed or proposed for Nm23 proteins. Nm23 proteins exhibit a nonspecific nucleoside diphosphate kinase (NDPK) 2The abbreviations used are: NDPKnucleoside diphosphate kinaseATXautotaxinawdabnormal wing discsawdk-pnawd killer of prunePAGEpolyacrylamide gel electrophoresis. activity (6Kimura N. Shimada N. Nomura K. Watanabe K. J. Biol. Chem. 1990; 265: 15744-15749Abstract Full Text PDF PubMed Google Scholar, 13Wallet V. Mutzel R. Troll H. Barzu O. Wurster B. Veron M. Lacombe M.L. J. Natl. Cancer Inst. 1990; 82: 1199-1202Crossref PubMed Scopus (223) Google Scholar, 44Biggs J. Hersperger E. Steeg P.S. Liotta L.A. Shearn A. Cell. 1990; 63: 933-940Abstract Full Text PDF PubMed Scopus (332) Google Scholar, 45Muñoz-Dorado J. Inouye M. Inouye S. J. Biol. Chem. 1990; 265: 2702-2706Abstract Full Text PDF PubMed Google Scholar). NDPK (EC2.7.4.6) catalyzes the transfer of a terminal phosphate of 5ʹ-triphosphate nucleotides to 5ʹ-diphosphate nucleotides via a high energy NDPK-phosphohistidine intermediate (46Krebs H.A. Hems R. Biochim. Biophys. Acta. 1953; 12: 172-180Crossref PubMed Scopus (90) Google Scholar, 47Berg P. Joklik W.K. Nature. 1953; 172: 1008-1009Crossref PubMed Scopus (49) Google Scholar, 48Parks R.E. Agarwal R.P. Nucleoside diphosphokinases. Academic Press, Inc., New York1973Google Scholar). The proposed biological functions of the NDPK activity include modulation of nucleotide pools, G-protein function, microtubule polymerization, etc. and have been subject to debate (reviewed in 36De La Rosa A. Williams R.L. Steeg P.S. BioEssays. 1995; 17: 53-62Crossref PubMed Scopus (225) Google Scholar). However, nm23 transfectants exhibiting suppressed metastatic potential did not have increased total or subcellular NDPK activity (49Golden A. Benedict M. Shearn A. Kimura N. Leone A. Liotta L.A. Steeg P.S. Nucleoside Diphosphate Kinase, nm23 and Tumor Metastasis: Possible Biochemical Mechanisms. Kluwer Academic Publishers, Boston, MA1993Google Scholar). Additionally, the killer of prune mutation of Drosophila awd (awdk-pn) (50Biggs J. Hersperger E. Dearolf C. Shearn A. Genes & Dev. 1988; 2: 1333-1343Crossref PubMed Scopus (123) Google Scholar) retained NDPK activity, yet caused all of the developmental abnormalities associated with null-awd when co-expressed with a prune (pn) mutation (51Lascu I. Chaffotte A. Limbourg-Bouchon B. Veron M. J. Biol. Chem. 1992; 267: 12775-12781Abstract Full Text PDF PubMed Google Scholar). Direct evidence indicating the necessity of the conserved histidine residue, but not for elevated NDPK activity, in Drosophila development was provided by transformation studies using nm23-H1, nm23-H2, and both wild type and histidine-mutated awd into the null-awd germ line (52Xu J. Liu L. Deng F. Timmons L. Hersperger E. Steeg P. Veron M. Shearn A. Dev. Biol. 1996; (in press)Google Scholar). A second biochemical activity for Nm23 proteins utilizing histidine phosphorylation as a histidine protein kinase was recently reported (53Wagner P. Vu N.-D. J. Biol. Chem. 1995; 270: 21758-21764Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar).Lower energy serine phosphorylation of Nm23, not directly involved in the phosphate transfers of the NDPK or histidine protein kinase activity, have also been described (54MacDonald N.J. De La Rosa A. Benedict M.A. Freije J.M.P. Krutzsch H. Steeg P.S. J. Biol. Chem. 1993; 268: 25780-25789Abstract Full Text PDF PubMed Google Scholar, 55Muñoz-Dorado J. Almaula N. Inouye S. Inouye M. J. Bacteriol. 1993; 175: 1176-1181Crossref PubMed Google Scholar, 56Hemmerich S. Pecht I. Biochemistry. 1992; 31: 4580-4587Crossref PubMed Scopus (44) Google Scholar, 57Bominaar A. Tepper A. Veron M. FEBS Lett. 1994; 353: 5-8Crossref PubMed Scopus (42) Google Scholar). For Nm23-H1, two proteolytic fragments containing serines 44 and 120, 122 and 125 exhibited serine autophosphorylation. In contrast to NDPK activity, serine phosphorylation levels among control- and nm23-transfected melanoma cell lines were directly correlated with metastasis-suppressive activity, suggesting the hypothesis that it may be biologically relevant (54MacDonald N.J. De La Rosa A. Benedict M.A. Freije J.M.P. Krutzsch H. Steeg P.S. J. Biol. Chem. 1993; 268: 25780-25789Abstract Full Text PDF PubMed Google Scholar).Other proposed biochemical activities for Nm23 proteins have been reported. Nm23-H2 has been identified as a transcription factor for the PuF site found near the c-myc and other promoters (58Postel E.H. Berberich S.J. Flint S.J. Ferrone C.A. Science. 1993; 261: 478-480Crossref PubMed Scopus (482) Google Scholar, 59Postel E.H. Ferrone C.A. J. Biol. Chem. 1994; 269: 8627-8630Abstract Full Text PDF PubMed Google Scholar), but recent data suggest that the DNA binding activity is nonspecific, including single- or double-stranded pyrimidines (60Hildebrandt M. Lacombe M. Mesnildrey S. Veron M. Nucleic Acids Res. 1995; 23: 3858-3864Crossref PubMed Scopus (70) Google Scholar). A phosphatase specific for Nm23 proteins, with homology to the Bax proteins involved in apoptosis, was recently described (61Shankar S. Kavanaugh-Black A. Kamath S. Chakrabarty A. J. Biol. Chem. 1995; 270: 28246-28250Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), suggesting complex regulatory interactions. Interaction of Nm23 proteins with cAMP have been described in vitro (54MacDonald N.J. De La Rosa A. Benedict M.A. Freije J.M.P. Krutzsch H. Steeg P.S. J. Biol. Chem. 1993; 268: 25780-25789Abstract Full Text PDF PubMed Google Scholar, 62Strelkov S. Perisic O. Webb P. Williams R. J. Mol. Biol. 1995; 249: 665-674Crossref PubMed Scopus (32) Google Scholar).We report herein site-directed mutagenesis of nm23-H1, assayed for effects on tumor cell motility in vitro. Our data provide the first link of Nm23 biochemical structure with its suppression of one component of the metastatic phenotype.RESULTSExpression of Transfected ConstructsMDA-MB-435 human breast carcinoma cells were transfected side by side with either a control pCMVBamneo (pCMV) vector, pCMVnm23-H1 or site-directed mutants of pCMVnm23-H1. The site-directed mutants are listed in Table I and include (a) nm23-H1S44-A, a serine to alanine alteration of amino acid 44, previously reported as a site of human Nm23-H1 phosphorylation (54MacDonald N.J. De La Rosa A. Benedict M.A. Freije J.M.P. Krutzsch H. Steeg P.S. J. Biol. Chem. 1993; 268: 25780-25789Abstract Full Text PDF PubMed Google Scholar); (b) nm23-H1P96-S, a proline to serine alteration of amino acid 96, the killer of prune mutation in the Drosophila Awd homolog of Nm23 (50Biggs J. Hersperger E. Dearolf C. Shearn A. Genes & Dev. 1988; 2: 1333-1343Crossref PubMed Scopus (123) Google Scholar); (c) nm23-H1S120-G, a serine to glycine alteration at amino acid 120, the most evolutionary conserved serine present on an Nm23-H1 acid-stable phosphorylated peptide (54MacDonald N.J. De La Rosa A. Benedict M.A. Freije J.M.P. Krutzsch H. Steeg P.S. J. Biol. Chem. 1993; 268: 25780-25789Abstract Full Text PDF PubMed Google Scholar), and a mutation found in 6 of 28 high grade human neuroblastomas (69Chang C. Zhu X. Thoraval D. Ungar D. Rawwas J. Hora N. Strahler J. Hansh S. Radany E. Nature. 1994; 370: 335-336Crossref PubMed Scopus (133) Google Scholar); (d) nm23-H1S120-A, a serine to alanine alteration at amino acid 120; (e) nm23-H1H118-F, a histidine to phenylalanine alteration at amino acid 118, the phosphorylated intermediate for the NDPK and possibly the histidine protein kinase reactions (48Parks R.E. Agarwal R.P. Nucleoside diphosphokinases. Academic Press, Inc., New York1973Google Scholar, 53Wagner P. Vu N.-D. J. Biol. Chem. 1995; 270: 21758-21764Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Approximately 30 colonies/transfection construct were harvested if available, with the exception of the control (empty vector) construct. Clones were initially screened for the overexpression of a nm23-H1 transcript on Northern blots. From these blots, 2-3 clones/transfection construct were selected as available for further expression, biologic, and biochemical analyses.Table I.Nm23-H1 site-directed mutant constructsConstructNumber of clones identifiedClones investigated for motilityNeomycin-resistantOverexpressing nm23-H1 mRNAControl140/14K10K27nm23-H18aIndicates total number of G418 colonies produced in transfection experiment. For other constructs, numerous colonies were produced, and approximately 30 were harvested for Northern analysis.5/8P52P57P62nm23-H1P96-S417/41S16S22S29bClones with minimal nm23-H1 mRNA overexpression but which exhibited no significant overexpression of Nm23-H1 protein on Western blots.nm23-H1S120-G327/32I108I205nm23-H1S120-A8aIndicates total number of G418 colonies produced in transfection experiment. For other constructs, numerous colonies were produced, and approximately 30 were harvested for Northern analysis.1/8R4nm23-H1S44-A2911/29J20J58nm23-H1H118-F6aIndicates total number of G418 colonies produced in transfection experiment. For other constructs, numerous colonies were produced, and approximately 30 were harvested for Northern analysis.2/6E2bClones with minimal nm23-H1 mRNA overexpression but which exhibited no significant overexpression of Nm23-H1 protein on Western blots.E6a Indicates total number of G418 colonies produced in transfection experiment. For other constructs, numerous colonies were produced, and approximately 30 were harvested for Northern analysis.b Clones with minimal nm23-H1 mRNA overexpression but which exhibited no significant overexpression of Nm23-H1 protein on Western blots. Open table in a new tab A Northern blot showing nm23-H1 mRNA levels in the clones selected from the transfection experiment is shown in Fig. 1A. Two control-transfected clones, K10 and K27, were randomly selected and exhibited uniformly low nm23-H1 mRNA expression. Three wild type nm23-H1-transfected clones (P52, P57, and P62) were selected with varying degrees of mRNA overexpression. At least two clones overexpressing nm23-H1 mRNA on the Northern blots were identified for each mutant construct, with the exception of nm23-H1S120-A, for which only one expressing clone was identifiable. Additionally, levels of nm23-H1 mRNA just above those of the control transfectants were observed in one of the only two expressing nm23-H1H118-F clones. Rehybridization of the Northern blot to a glyceraldehyde-3-phosphate dehydrogenase probe demonstrated equivalent loading of RNA (Fig. 1B). Further biochemical and biologic analyses of the transfectants proceeded in parallel after Northern blot hybridization experiments.Fig. 2 presents a Western blot analysis of Nm23 protein levels in each of the transfectants. The wild type (P) transfectants expressed 2.1-4.1-fold greater Nm23-H1 than the mean level of the control (K) transfectants, as determined by densitometry of chemiluminescence exposures. At amino acid 96, two of the Nm23-H1P96-S (S) clones expressed significantly greater Nm23-H1 than the control (K) clones. The S29 clone, which exhibited nm23-H1 RNA overexpression, expressed little additional Nm23-H1 protein when blots were normalized by cell number (Fig. 2) or by micrograms of protein in cell lysates (data not shown). At amino acid 120, two Nm23-H1S120-G (I) clones as well as the single Nm23-H1S120-A (R) clone expressed 1.6-3.1-fold greater Nm23-H1 than the control (K) transfectants. For amino acid 44, the J20 transfectant of the Nm23-H1S44-A construct expressed 3-fold more protein than the control (K) transfectants. Interpretation of the J58 transfectant data at the same amino acid position is difficult. Whereas this clone overexpressed nm23-H1 mRNA, it exhibited little additional Nm23-H1 protein in Western blots normalized by cell number (Fig. 2). When normalized by micrograms of protein in the cell lysate, the J58 clone exhibited 2.1-fold Nm23-H1 overexpression relative to the control (K) transfectants (data not shown). The data suggest that expression of the transfected cDNA was obtained in this clone but was accompanied by additional changes in cell size, protein synthetic rates, etc. For the histidine 118 position, the E6 transfectant of the Nm23-H1H118-F construct expressed 6.7-fold greater Nm23-H1 than control (K) transfectants. The E2 clone, which expressed little additional nm23-H1 mRNA on Northern blots, failed to significantly overexpress Nm23-H1 when Western blots were normalized by either cell number (Fig. 2) or micrograms of protein in lysates (data not shown).Fig. 2Western blot of Nm23-H1 protein levels of control-, wild type nm23-H1-, and site-directed nm23-H1 mutant-transfected clones. MDA-MB-435 human breast carcinoma cells were transfected with each construct, and clones were harvested as indicated in Table I. Lysate from 3 x 106 cells of each transfectant was electrophoresed in a 14% SDS-PAGE, which was processed as a Western blot using affinity-purified anti-Nm23 peptide 11 antibody (2Rosengard A.M. Krutzsch H.C. Shearn A. Biggs J.R. Barker E. Margulies I.M.K. King C.R. Liotta L.A. Steeg P.S. Nature. 1989; 342: 177-180Crossref PubMed Scopus (478) Google Scholar). The mobilities of Nm23-H1 and Nm23-H2 proteins are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In Vitro Motility of Site-directed MutantsFetal Calf SerumMotility was determined in Boyden chamber assays using fetal calf serum or partially purified ATX as a chemoattractant. Serum was selected for two reasons: (a) clones of murine K-1735 TK melanoma transfected with murine nm23-1 or human MDA-MB-435 breast carcinoma transfected with human nm23-H1 exhibited reduced motility responses to serum (42Kantor J.D. McCormick B. Steeg P.S. Zetter B.R. Cancer Res. 1993; 53: 1971-1973PubMed Google Scholar), and (b) serum contains a variety of motility stimulating and inhibiting factors, which may better reflect the milieu to which tumor cells are exposed in vivo. Each cell line was assayed in triplicate with 0, 0.25, 0.5, and 1.0% (v/v) serum, for which peak responses were typically obtained at 0.5%. Most experiments tested a subset of the total number of transfected cell lines, due to strict culture requirements for reproducible assays, the number of Boyden chambers available, and potential deleterious effects of long times involved in handling and counting multiple cell lines. All experiments included at least one control transfectant (K) and one wild type nm23-H1 transfectant (P). Table II lists data from three successive experiments, showing motility of cells from the various clones to 0.5% serum. For experiment 481, both control-transfected (K) clones migrated toward serum, with 17-23 cells/high power field. Expression of wild type nm23-H1 by the P52, P57, and P62 clones resulted in a 44-98% reduction in migration, calculated on the basis of the mean of the K clones. The migration of P52 exhibited variability between experiments, whereas that of the P57 and P62 clones was uniform. Reduced motility was exhibited by the P57 clone in experiments 482 and 483.Table II.In vitro motility of MDA-MB-435 human breast carcinoma cells transfected with control, wild type, or site-directed mutant constructs toward fetal calf serumTransfection constructMean cells migrating to 0.5% serum ± S.E.aCells were tested in Boyden chambers for in vitro motility to 0.25, 0.50, 0.75, and 1.0% (v/v) fetal calf serum, in 8-h assays. The mean ± S.E. cells migrating through a type IV collagen-coated 8-μm filter toward serum, determined from counting six to nine x 40 microscopic fields per Boyden chamber, three chambers per data point, were calculated.Expt. 481Expt. 482Expt. 483ControlK1023.36 ± 0.339.78 ± 3.920.34 ± 2.8K2717.84 ± 0.4Nm23-H1P5211.42 ± 5.5P571.00 ± 0.71.54 ± 0.61.54 ± 0.4P620.42 ± 0.3Nm23-H1P96-SS1610.50 ± 1.2162.0 ± 1.2S2241.84 ± 6.523.33 ± 3.6S2917.50 ± 3.757.56 ± 4.2Nm23-H1S120-GI10842.17 ± 6.6114.3 ± 8.5I20543.42 ± 3.262.75 ± 3.2Nm23-H1S120-AR41.08 ± 0.82.00 ± 1.7Nm23-H1S44-AJ2011.17 ± 0.6J581.00 ± 0.615.11 ± 1.5Nm23-H1H118-FE244.42 ± 7.688.28 ± 3.2E60a Cells were tested in Boyden chambers for in vitro motility to 0.25, 0.50, 0.75, and 1.0% (v/v) fetal calf serum, in 8-h assays. The mean ± S.E. cells migrating through a type IV collagen-coated 8-μm filter toward serum, determined from counting six to nine x 40 microscopic field" @default.
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• W2000002361 title "Site-directed Mutagenesis of nm23-H1" @default.
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