The activation of the heme oxygenase (HO), fos, and AP-1 genes was determined at intervals after treatment of M1 cells (derived from mouse myeloleukemia) with heme or 12-O-tetradecanoylphorbol 13-acetate (TPA). On treatment with heme, M1 cells did not differentiate into macrophage but their HO gene was activated and showed maximum transcriptional activity after treatment with heme for 2 h. In contrast, on treatment with TPA, these cells adhered to the culture flasks within 5 h and differentiated into macrophage-like cells. Moreover, the sequential activation of the fos, AP-1, and HO genes was observed during this period, with the maximal transcriptional activities after TPA treatment for 0.5, 1, and 1.5 h, respectively. Thus, the HO gene was activated by treatment with either heme or TPA, and the latter activation was associated with activations of the fos and AP-1 genes. As the rat HO gene is known to have a TPA-sensitive element in its promoter region, this gene was suggested to be activated by a fos-AP-1 complex protein.
Using DAPI, rabbit antitubulin antibody, FITC-labeled goat anti-rabbit IgG, and TRITC-phalloidin to stain individual celIs, the microspectrophotometric analysis showed that three markers that represent the nucleus, microtubules (MT), and microfilaments (MF), respectively, could be recognized in individual cells without interference. The phase of the cell cycle was determined by DNA content. We found that in Indian muntjac (IM) cells, the amount of tubulin in Cz and M phases was about twice as much as that in G1 phase. In G2 cells, the cytoplasmic microtubule complex (CMTC) became denser than in GI cells. The cytoplasmic MT extent in basically the same orientation as MF bundles in interphase. The regions where the MT is denser also have a denser MF distribution.Key terms: Microspectrophotometry, stress fiber, cytoplasmic microtubule complexThe structure and distribution of microtubules (MT) and microfilaments (MF) have regulating roles in the cytophysiological process for many cells (6). Cellular morphology varies in different phases of the cell cycle (21,22). The depolymerization of MT induced by growth factors or oncogenic viruses induces the Go cells to enter S phase (1,9,10,26). Dynamic changes in several cytoskeletal compartments are closely related to cellular morphology and function.According to some reports, there is no obvious change in actin content during the cell cycle (13,251. Tubulin synthesis, however, whether in lower or higher biological species, is quite distinct in various cycle phases. I n physarum and chlamydomonas, tubulin and its mRNA synthesis increase rapidly and reach peak values in M phase (14,171. In HeLa and CHO cell lines, tubulin synthesis increases gradually from S phase. The amount of tubulin reaches a maximum in Gg phase and is maintained at this level in M phase (5,241. The results mentioned above were all obtained from the synchronized cells. However, in mammalian cells it is fairly difficult to obtain a pure population of synchronized cells, especially in Gz phase. In our report, three fluorescent dyes were used to stain DNA, MT, and MF in the same cell. Cell cycle phase is identified by DNA content. With this method we could observe and detect tubulin content and the distribution of MT and MF in different cell cycle phases and research the cytoskeleton characteristics in the cell cycle. washed twice with PBS, and air-dried. The fixed cells were incubated with antitubulin antibody for 60 rnin at 37°C. Then the coverslips were thoroughly washed with PBS, 1% Triton X-IOOPBS, and PBS in turn each for 5 min, air-dried, incubated with FITC-goat anti-rabbit IgG antibody (Beijing Institute of Biological Reagents) for 60 min at 37"C, and then washed as described above. The MT-stained coverslips were immersed in -20°C acetone for 3-5 min and then dried. Subsequently, the cells were incubated for 20 min at room temperature with TRITC- measured with a D filter block consisting of blue excitation filter (450-580-nm bandpass filter), RKP 510 dichroic mirror, and high built-in barrier filt...
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