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Calcium, cyclic AMP, and cyclic GMP do not seem to be involved in proliferative activation of postmitotic differentiated cells. Instead, they are intracycle regulators, and we propose the following working model of their control of the initiation of DNA synthesis. While a role for cyclic GMP cannot yet be defined, a brief postmitotic burst of its synthesis might serve to prevent certain activated cells (e.g. 3T3 mouse cells) from being diverted into a nonproliferating (but still activated) G0 state (Figs. 1 and 17). In a latter part of the G1 phase, something happens to stimulate briefly the synthesis of cyclic AMP which, in turn, drives calcium ions from the mitochondria into the cytosol to activate newly synthesized thymidylate synthetase (or other primed enzymic assemblies) (Fig. 1). Having "turned on" their target enzymes, the accumulated cyclic AMP is destroyed and the excess calcium ions are reaccumulated by the mitochondria to avoid interfering with succeeding reactions. This model predicts that persistent changes in cyclic AMP metabolism and the respiration-linked, calcium-accumulating (ion-buffering) activity of mitochondria may be responsible for the sustained growth of tumors.

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In recent studies on the cytoskeletal organization of T51B rat liver cells by indirect immunofluorescence microscopy, we have been unable to achieve double-staining of microtubules and intermediate filaments within the same cell. In acetone-fixed cells, microtubules were poorly preserved, and two out of three monoclonal antibodies tested did not stain them properly. In formaldehyde-fixed cells, the monoclonal anti-cytokeratin produced an incomplete staining pattern against a diffuse background. We have now developed a fixation protocol which includes simultaneous fixation and extraction with formaldehyde and nonionic detergent in the present of microtubule stabilization buffer. Although developed for a specific purpose, it is of general application as it yields excellent preservation of all cytoskeletal components tested so far, without masking antigenic determinants. The procedure is both simple and fast and will, therefore, be valuable for efficient processing of samples from large-scale experiments, such as the screening for cytoskeletal changes during longterm treatment of cells with drugs or carcinogens.

Mouse and human extracted liver tissue were examined by indirect immunofluorescent staining and transmission electron microscopy in order to study the alteration of cytokeratin intermediate filaments associated with Mallory body formation. Frozen sections of griseofulvin-fed mouse liver and human liver of primary biliary cirrhosis and primary sclerosing cholangitis were extracted by Triton X-100 and nuclease. Indirect immunofluorescent staining was performed by using anticow hoof keratin antibody for mouse liver and anti-human epidermal keratin antibody (AE1 and AE3) for human liver. Transmission electron microscopy was also performed on extracted and critical point-dried samples. The griseofulvin-fed mouse hepatoma cells showed four different types of altered staining pattern based on the indirect immunofluorescent staining of the cytoplasm and Mallory bodies: Type I--cytoplasm(+), Mallory body(-); Type II--cytoplasm(+), Mallory body(+); Type III--cytoplasm(-), Mallory body(+), and Type IV--cytoplasm(-), Mallory body(-). Types I and III were predominant, however, some hepatoma cells which contain Mallory bodies revealed bright cytoplasmic staining (Type II). The nuclear rims were strongly stained. In human liver, AE1 stained Mallory bodies and the bile duct epithelium intensely, but did not stain normal hepatocytes. AE3 mainly stained Mallory bodies and normal hepatocytes, but also stained bile duct epithelium weakly. Indirect immunofluorescent staining for human liver showed the same staining patterns as found in mouse liver, except that Type IV was not observed. Although many hepatocytes which contained Mallory bodies did not react with either of these two antibodies (Type III), some of the hepatocytes were stained, not only with AE3, but also with AE1 (Type II).(ABSTRACT TRUNCATED AT 250 WORDS)