As similarly observed in nutrient-poor media at 37∞C, Escherichia coli forms small rods in nutrient-rich media at temperatures near 8∞C, the minimum temperature of growth. A study was initiated to identify proteins required to facilitate the small rod morphology at low temperature. E. coli contains three nonessential SPOR domain proteins (DamX, RlpA, and DedD) that have been demonstrated to bind to the septal ring. In contrast to the normal growth and small rod morphology of damX and rlpA null mutants at 10∞C, the dedD null mutant exhibited reduced growth and formed filamentous cells. The presence of plasmid-encoded DedD restored growth and small rods. Plasmid-encoded FtsN, an essential SPOR domain protein that functions to stabilize the septal ring and to initiate septation, in the dedD null mutant resulted in increased growth and the formation of shorter chained cells. However, plasmid-encoded DedD failed to restore growth and cell division of cells lacking FtsN at 10∞C. In contrast to cell division protein DedD, RodZ is a cell elongation protein particularly required for growth at 30∞C. However, the rodZ null mutant grew similarly as the wild type strain and produced cocci in LB broth at 10∞C. Moreover at 10∞C, the concerted deletion of dedD and rodZ resulted in severe inhibition of growth accompanied with the formation of swollen prolate ellipsoids due to a block in septal ring assembly and cell elongation. The data indicate the cellular requirement of both FtsN and DedD for septation as well as RodZ for cell elongation to maintain the small rod morphology at temperatures near 8∞C.In comparison to the growth and small rods of the wild type in M9-glucose minimal media at 37∞C, the dedD null mutant grew at the same rate and produced elongated cells while the rodZ null mutant grew at a slightly slower rate and produced cocci. The data indicate that DedD and RodZ are also required to maintain the small rod morphology in nutrient-poor media, but there is a higher cellular requirement of DedD for growth and cell division in nutrient-rich media at low temperature.

Purpose: The aims of this study were to investigate the correlation between MAPK activation and somatic mutation in GNAQ and GNA11 genes in uveal melanoma (UM) and to investigate the potential utility of MEK inhibition as a target for therapy of UM in tumors with GNAQ and GNA11 mutations. Methods: Sequencing and restriction fragment length polymorphism (RFLP) were utilized for detection of activating mutations in codon 209 of the GNAQ and GNA11 genes in 44 primary UMs. The expression of phospho p44/42 MAPK (pERK1/2) was assessed by immunohistochemistry in 44 UMs. In addition, the expression of pERK1/2 and pMEK 1/2 were studied using Western blot analysis in 17 primary UMs (14 with GNAQ or GNA11 mutations) and five UM cell lines (C918, 92.1, OCM3, MEL202, MEL270). Three of the UM cell lines (92.1, MEL202 and MEL270) have GNAQ codon 209 mutations. The effect of three different selective MEK inhibitors (U126, PD98059 and PD184352) on UM cell proliferation and apoptosis were studied. Results: The majority (30/44, 68.2%) of the primary UM tumors showed somatic mutation in codon 209 of either the GNAQ or the GNA11 genes. With the exception of one tumor, the mutations in either gene were mutually exclusive. Of the 30 UM primary tumors with GNAQ and/or GNA11 mutation, pERK1/2 expression was absent in 26.7%, weak in 33.3%, moderate in 23.3%, and high in only 16.7%. Western blot showed no pMEK1/2 expression in 6/14 (42.9%) of UM with either GNAQ or GNA11 mutations. Two of the UM cell lines with GNAQ mutation showed weak pERK1/2 expression. In addition one showed no pMEK1/2 expression while the other one showed very weak pMEK1/2 expression. Selective MEK inhibitors slowed UM cell proliferation but didn't induce significant apoptosis in UM cell lines regardless of GNAQ status. Conclusions: Our results indicate that a significant number of UM with somatic mutations in GNAQ or GNA11 showed no MAPK pathway activation. In UM, MEK inhibition is mostly cytostatic suggesting that selective MEK inhibitors alone may not be sufficient to control UM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1127. doi:10.1158/1538-7445.AM2011-1127