KRAS mutations occur in one third of human cancers and cluster in several hotspots, with codons 12 and 13 being most commonly affected. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. We used MCF10A human mammary epithelial cells to establish isogenic cell lines that express different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological or elevated levels, and investigated the biochemical and functional consequences of the different variants. The overall effects of low-expressing mutants were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes, migratory abilities, or increased phosphorylation of ERK, PDK1, and AKT. KRAS-G12D, G12V, G13D, and K117N mediated EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by K117N and Q61H. Both codon 13 mutations were associated with increased EGFR expression. Finally, global gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells.

Summary. An in vitro assay is presented in which different soluble substrates are arranged in narrow alternating stripes which forces growing axons and migratory cells to choose between them. The usefulness of this assay is exemplified by offering goldfish retinal axons and glial cells of the optic nerve a variety of substrates in stripes. Given a choice between substrates of unequal growth supporting activities axons and migratory cells grow in stripes, thus expressing their preference for one of the substrates. Growth in stripes was observed 1. when a substrate with growth promoting properties was next to one which did not possess these properties, 2. when the growth promoting activity of a substrate applied to both stripes was in one stripe blocked by an antibody, 3. when two different growth promoting substrates were offered.

In order to test the preference of growing axons for membrane-associated positional specificity a new in vitro assay was developed. In this assay, membrane fragments of two different sources are arranged as a carpet of very narrow alternating strips. Axons growing on such striped carpets are simultaneously confronted with the two substrates at the stripe borders. If there is a preference of axons for one or the other substrate they become oriented by the stripes and grow within the lanes of the preferred substrate. Such preferential growth could, in principle, be due to affinity to attractive factors on the preferred stripes or avoidance of repulsive factors on the alternate stripes. This assay system was used to investigate growth of chick retinal axons on tectal membranes. Tissue strips cut from various areas of the retina were explanted and the extending axons were confronted with stripes of cell membranes from various areas within the optic tectum. Tectal cell membranes prove to be an excellent substrate for the growth of retinal axons. Nasal and temporal axons can grow well on membranes of both posterior and anterior tectal cells. If, however, temporal axons are given a choice and encounter the border between anterior and posterior membranes they show a marked preference for growth on membranes of the anterior tectum, their natural target area. Nasal axons do not show a preference in this assay system. The transition from nasal to temporal properties within the retina is abrupt. In contrast, the transition from anterior to posterior properties of the tectal cell membranes occurs as a smooth gradient. Significantly, the positional differences of tectal membrane properties are only seen during the period of development of the retinotectal projection and are independent of tectal innervation by retinal axons. These anterior-posterior differences disappear by embryonic day 14.

Background: Translational regulation might underlie the high expression levels of the protease cathepsin L (CTSL) associated with poor breast cancer prognosis. Results: Translation of CTSL mRNA is highly stress-resistant and promotes metastasis of murine breast cancer. Conclusion: CTSL mRNA circumvents translational shutdown in cancer-associated stress conditions. Significance: High expression of a metastasis promoting protease is maintained by translational regulation.