Sequence and expression of the kettin gene in Drosophila melanogaster and Caenorhabditis elegans.

Kettin is a large modular protein associated with thin filaments in the Z-disc region of insect muscles. The sequence of a 21.3 kb contig of the Drosophila gene has been determined. The corresponding protein sequence has 35 immunoglobulin-like (Ig) domains which are separated by shorter linker sequences, except near the N and C termini of the molecule where linker sequences are short or missing. This confirms a model in which each Ig domain binds to an actin protomer. The Drosophila kettin gene is at 62C 1-3 on the third chromosome. Two P-element insertions, l(3)j1D7 and l(3)rL182 are in the kettin gene, and complementation tests showed that existing l(3)dre8 mutations are in the same gene. The RNA was detected in wild-type Drosophila embryos at stage 11, first in the gut invagination region of the mesoderm, and by stage 13 in both visceral and somatic mesoderm. Somatic mesoderm expression became segmental at stage 13. RNA expression was greatly reduced in embryos of P-element homozygotes but normal in heterozygotes. The structure of the flight muscle in all the heterozygous mutants was normal, including the myofibril-cuticle connections, and they were able to fly. Kettin sequence homologous to the Drosophila protein, was identified in the Caenorhabditis elegans genome database. The RNA was detected in pharyngeal, body wall and anal depressor muscles of larvae and adult worms, as well as in the male gonad. Antibody to insect kettin labelled the pharyngeal, body wall, anal depressor and proximal gonadal muscles in adult worms. Body wall muscles were labelled in an obliquely striated pattern consistent with the Z-disc localisation in insect muscle. The relationship of kettin to D-titin, which has been assigned to the same chromosomal locus in Drosophila, is discussed.

A microplate-based fluorometric assay for monitoring human cancer cell attachment to cortical bone.

A microplate-based fluorometric assay was developed for quantitation of cancer cell attachment to bone matrix. To evaluate this model system we used a panel of human cancer cell lines, including the human breast cancer cell line MDA-MB-231. For the assay, bovine cortical bone from the shaft of the femur was cut, turned, and sliced to 6-mm-diameter round 200-microm-thin disks and placed into the wells of a 96-well microplate. A fluorescent dye 2', 7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein (BCECF-AM) and a microplate fluorometer equipped with a standard filter set for fluorescein isothiocyanate were used to detect the cells attached to the bone disks. The fluorescence was enhanced during the measurement step by using a K+ solution (pH 8.0) containing the H+/K+ ionophore nigericin, which equilibrates internal and external pH. By taking advantage of the pH sensitivity of BCECF, the fluorescence intensity in the assay was increased 2.5 times compared to cells loaded with calcein. The specificity of the assay was demonstrated with a specific immuneserum raised against the MDA-MB-231 cell line. The assay can be completed in 1 h and permits the use of a large number of samples and is therefore useful for screening potential drug candidates that could block cancer cell attachment to bone material. Moreover, the assay described can easily be used to characterize molecular structures involved in cell attachment to bone.