Cyclin D1 as a proliferative marker regulating retinoblastoma phosphorylation in mouse lung epithelial cells.

Elevations in cyclin D1 content increase the phosphorylation status of retinoblastoma (Rb) protein to encourage cell cycle transit. We sought to determine if cyclin D1 content could be used as an index of cell proliferation in mouse lung epithelia following growth manipulations in vitro and in vivo. Rb protein concentration was high in 82-132 and LM2, two fast-growing neoplastic mouse lung epithelial cell lines. The hyperphosphorylated form of Rb predominated in these two cell lines, while Rb in slower-growing cell lines was predominantly hypophosphorylated. Consistent with this, more cyclin D1 protein was expressed in the fast-growing cell lines than in slower-growing cells. We therefore tested whether cyclin D1 content varied with growth status. The amount of cyclin D1 decreased upon serum removal coincident with growth inhibition and then increased upon serum re-addition which stimulated resumption of proliferation. This correlation between cyclin D1 content and growth status also occurred in vivo. Cyclin D1 content increased when lungs underwent compensatory hyperplasia following damage caused by butylated hydroxytoluene administration to mice and in lung tumor extracts as compared with extracts prepared from uninvolved tissue or control lungs. We conclude that elevated cyclin D1 levels account, at least in part, for the hyperphosphorylation of Rb in neoplastic lung cells, and are associated with enhanced lung growth in vitro and in vivo.

Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle.

The three dimensional organization of microtubules in mitotic spindles of the yeast Saccharomyces cerevisiae has been determined by computer-aided reconstruction from electron micrographs of serially cross-sectioned spindles. Fifteen spindles ranging in length from 0.6-9.4 microns have been analyzed. Ordered microtubule packing is absent in spindles up to 0.8 micron, but the total number of microtubules is sufficient to allow one microtubule per kinetochore with a few additional microtubules that may form an interpolar spindle. An obvious bundle of about eight interpolar microtubules was found in spindles 1.3-1.6 microns long, and we suggest that the approximately 32 remaining microtubules act as kinetochore fibers. The relative lengths of the microtubules in these spindles suggest that they may be in an early stage of anaphase, even though these spindles are all situated in the mother cell, not in the isthmus between mother and bud. None of the reconstructed spindles exhibited the uniform populations of kinetochore microtubules characteristic of metaphase. Long spindles (2.7-9.4 microns), presumably in anaphase B, contained short remnants of a few presumed kinetochore microtubules clustered near the poles and a few long microtubules extending from each pole toward the spindle midplane, where they interdigitated with their counterparts from the other pole. Interpretation of these reconstructed spindles offers some insights into the mechanisms of mitosis in this yeast.