Novel nuclear and cytoplasmic proteins detected by anti-Zoothamnium arbuscula (Protozoa) spasmin 1 antibody in mammalian cells are dependent on the cell cycle.

Spasmin is a calcium-binding protein that is the major component of calcium-induced contractile filaments, called spasmoneme, found in vorticellid ciliates. Such filaments have not been observed in any organisms other than green algae. To determine whether calcium-induced contractile filaments resembling spasmoneme are present in higher eukaryotes, we performed immunofluorescence imaging of an anti-Zoothamnium arbuscula (protozoa, ciliophora) spasmin 1 polyclonal antibody in HeLa cells. In the cytoplasm, ubiquitous antigens seemed to be co-localized with microtubules at interphase, but not throughout mitosis. In the nucleus, areas linked to the nuclear envelope contained a number of hot spots. These regions were unclear during condensation of the replicated chromosomes, but became clearly visible again at cytokinesis. Immunoblotting analysis identified localized antigens during different phases of the cell cycle, including a 68/71 kDa cytoplasmic protein and a 55 kDa nuclear protein in interphase, and a 55/70 kDa protein in mitosis. The anti-spasmin 1 antibody recognized antigens in both hamster kidney BHK21 cells and Human lung cancer A-549 cells. These results suggest that novel spasmin-like proteins could be common in mammalian cells.

Immunocytochemical expression of microtubule-associated protein-2 (MAP-2) in small cell lung cancer cell lines with neuronal-like processes.

Five of six human small cell lung cancer (SCLC) cell lines changed morphologically into cells with neuronal-like processes on the extracellular matrix of human lung adenocarcinoma cell line PC-9 cells (PC-9 / ECM substrate). The features of the neuronal-like processes of these SCLC cell lines were examined immunocytochemically using monoclonal antibodies against beta-chains of tubulin and microtubule-associated protein-2 (MAP-2), which is somatodendritic MAP of neurons. It was observed that beta-chains of tubulin and MAP-2 were expressed along the neuronal-like processes of SCLC cell lines. These findings suggest that the beta-chains of tubulin and MAP-2 are expressed functionally in SCLC cell lines in association with the development of dendrite-like processes on PC-9 / ECM substrate.

A novel target gene, SKP2, within the 5p13 amplicon that is frequently detected in small cell lung cancers.

We investigated DNA copy-number aberrations in 22 cell lines derived from small cell lung cancers (SCLCs) using comparative genomic hybridization. A minimal common region at 5p13, within the 5p11-p13 amplicon that was most frequently involved, harbored the CDH6, PC4, and SKP2 genes. These three genes showed amplification and consequent overexpression in the SCLC cell lines. SKP2 positively regulates progression of cell cycle by targeting several regulators, such as the cell-cycle inhibitor p27(KIP1), for ubiquitin-mediated degradation. SKP2 was amplified in 7 (44%) of 16 primary SCLC tumors, and consequently overexpressed in 10 (83%) of the 12 of those tumors we examined. Expression levels of SKP2 protein were cell cycle-dependent in SCLC cells as well as in normal cells, and were correlated with the DNA copy-number of the gene. There was an inverse correlation between the expression of SKP2 and p27(KIP1) proteins. Down-regulation of SKP2 using an anti-sense oligonucleotide remarkably suppressed the growth of SCLC cells. Our results indicate that SKP2 is likely to be a target of the 5p13 amplification and to play an important role in the growth of SCLC cells.