We localized the methionine aminopeptidase (map) gene on the Escherichia coli chromosome next to the rpsB gene at min 4. Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-p-thiogalactoside. Thus, methionine aminopeptidase is essential for cell growth.The map gene of Escherichia coli encodes the methionine aminopeptidase that preferentially removes the initiation methionine from certain intracellular proteins in vitro and in vivo (3). The substrate specificity of the methionine aminopeptidase is primarily determined by the residue adjacent to the methionine (3,5,11). The map location of the map gene and the biological significance of selective removal of the initiation methionine were not known. We report here the localization of the map gene on the E. coli genetic map and data indicating that methionine aminopeptidase performs an essential function in E. coli.The coding and flanking nucleotide sequences of the map gene have been reported elsewhere (3). We searched the GenBank DNA data base for E. coli sequences that matched the upstream sequence of map and identified the first 134 base pairs of the reported 5'-flanking sequence of the rpsBtsf operon (ribosomal protein S2 and translation elongation factor EF-Ts genes) (1). Thus, map is located 357 base pairs from the rpsB-tsf operon, and the two genes are transcribed divergently. The rpsB gene maps about 5 kilobases clockwise from the dapD gene at the 4-min position on the E. coli chromosome (2,4,8). The order of the four closely clustered genes is dapD-map-rpsB-tsf (Fig. IA). This conclusion is consistent with that deduced independently from the genomic restriction mapping data (9).We attempted by gene replacement, but failed, to substitute the chromosomal map+ gene with an inactivated copy of map (unpublished data). This led us to speculate that map is essential. Therefore, we substituted the chromosomal map+ gene with an altered map gene fragment that allowed us to conveniently manipulate its expression. We deleted the lacZ promoter sequence between the PvuII site and the EcoRI site in the pUC18 (13) (Fig. 1B), contains the map gene controlled by the lac promoter and operator fragment (lacZplo-map), the cat gene as a selectable marker, and flanking homologous sequences to allow double recombination for gene replacement.Plasmid pSYC1695 DNA was digested with Asp718 and SalI restriction enzymes, which cleave outside of the catlacZplo-map fragment. The DNA fragment was used to transform E. coli H205, an hsdR derivative from the recB recC sbcB strain JC7623 (12) that allows gene replacement through double homologous recombination between the linear transforming DNA and the chromosome (Fig. 1). Chloramphenicol-resistant (Cmr) transformants were selected on plates supplemented with the lac operon inducer isopropyl-,-thiogalactoside (IPTG) to induce and maintain the Map' phenotype in the transformants. One of these (designated EM1) was selected for further characterizati...

© 2 0 0 2 L a n d e s B i o s c i e n c e . N o t f o r d i s t r i b u t i o n .[Cancer Biology & ABSTRACTCellular adhesion molecules of the cadherin, integrin, and immunoglobulin superfamilies are important to both growth and metastasis of many cancers, including malignant melanoma. Malignant melanoma is an excellent model for studying these molecules, due in part to a sequential series of five defineable stages. As the malignant phenotype of melanoma cells changes from the noninvasive radial growth phase to the vertical growth phase, which has high metastatic potential, so does the repertoire of the cellular adhesion molecules expressed on the cells surface. The cellular adhesion molecule MCAM/ MUC18 confers metastatic potential and increased tumorgenicity to melanoma cells. MCAM/MUC18 mediates homotypic and heterotypic adhesion between melanoma cells and endothelial cells, respectively. Both types of interaction may promote metastasis at different stages in the metastasis cascade. We developed a fully humanized antibody to MCAM/MUC18 (ABX-MA1) that blocked melanoma metastasis in vivo. Furthermore, ABX-MA1 blocked the homotypic interaction between melanoma cells and endothelial cells as well as the promoter and collagenase activity of MMP-2. During melanoma progression the loss of E-cadherin expression disrupts normal homeostasis in the skin by freeing melanoma cells from structural and functional regulation by keratinocytes. The loss of functional E-cadherin is paralled by a gain in N-cadherin function that mediates homotypic interaction between melanoma cells, facilitates gap-junctional formation with fibroblasts and endothelial cells and promotes melanoma cell migration and survival. In addition, loss of E-cadherin may affect the β-catenin/wnt signaling pathways, resulting in deregulation of genes involved in growth and metastasis. The integrin family member α v β 3 is widely expressed on melanoma cells in the vertical growth phase. When α v β 3 is expressed in melanoma cells in the radial growth phase, this integrin is associated with increased tumor growth in vivo. α v β 3 may also promote melanoma invasion, through an interaction with MMP-2, and transendothelial migration, via a heterotypic melanomaendothelial cell interaction. This review summarizes recent knowledge on how changes in these adhesion molecules contribute to the acquisition of the metastatic phenotype in human melanoma.

Our results have significant clinical implications. Treatment of melanoma patients with dacarbazine could select for a more aggressive melanoma phenotype. We propose that combination treatment with anti-VEGF/IL-8 or MEK inhibitors may potentiate the therapeutic effects of dacarbazine.

Objective: Lung-MAP (SWOG S1400) is a master platform trial assessing targeted therapies in squamous NSCLC. The objective of study C (S1400C) was to evaluate the response rate to palbociclib, a cyclin-dependent kinase 4 and cyclin-dependent kinase 6 inhibitor, in patients with cell cycle gene abnormalities. Methods: Patients with squamous NSCLC, a performance status of 0 to 2, and normal organ function who had progressed after at least one prior platinum-based

Platelet-derived growth factor (PDGF) and its cognate receptor are widely expressed on melanomas. Coexpression of the growth factor and receptor suggests their role in autocrine or paracrine growth mechanisms. Imatinib mesylate was previously reported to have specific activity in inhibiting select tyrosine kinase receptors, including PDGF and c-Kit. Melanoma cells express abundant levels of the PDGF receptor (PDGFR). Nevertheless, c-Kit expression is progressively lost as the cells take on a more highly metastatic phenotype. To investigate the potential of imatinib mesylate as a therapy for melanoma, we studied its effect on the growth of melanoma cells using an in vivo mouse model. Melanoma cells with high malignant potential (PDGFR-positive, c-Kit-negative) or low malignant potential (PDGFR-positive, c-Kit-positive) were injected subcutaneously into athymic nude mice. Mice were treated with imatinib mesylate (100 mg/kg three times weekly) or with phosphate-buffered saline for 4 to 6 wk. PDGFR-alpha and -beta were expressed on all melanoma cell lines tested. The level of PDGFR expression correlated with the metastatic potential of the melanoma cells: higher levels of PDGFR-alpha were expressed on cells with higher metastatic potential, and higher levels of PDGFR-beta were expressed on cells with lower metastatic potential. There was no significant difference in tumor size between treated and control mice. Immunohistochemical studies demonstrated inhibition of PDGFR phosphorylation on the tumors from mice treated with imatinib mesylate but not from control mice, suggesting that the receptors were functional and that the concentration of drug used was appropriate. Our data demonstrated that imatinib mesylate blocked both PDGFR-alpha and PDGFR-beta in vivo. It did not, however, affect the growth of melanoma cells expressing PDGFR, regardless of whether the cells expressed c-Kit.