Fine Structural Alterations in Cell Particles During Chemical Carcinogenesis : II. Further Evidence for Their Involvement in the Mechanism of Carcinogenesis. The Swelling of Rat Liver Mitochondria During Feeding of Amino Azo Dyes

Swelling under carefully controlled conditions has been used to study alterations in the structure of rat liver mitochondria as a result of feeding azo dyes. The changes of the swelling properties of the mitochondria during feeding of the hepatocarcinogenic 3'-methyl-4-dimethylaminoazobenzene are essentially comparable to those observed previously with the microsomes, under the same dietary conditions. These alterations in mitochondrial swelling are not related to changes in the amount of these cell particulates per unit weight of tissue, during feeding of this azo dye. As with the microsomes, feeding of the isomeric but relatively noncarcinogenic 2-methyl-4-dimethylaminoazobenzene does not affect swelling. The structural differences between liver and hepatoma mitochondria show up not only in the rate and extent of swelling but also in the form of the curves of pH dependence. The influence of ketones and sulfhydryl compounds on the swelling of normal liver mitochondria were studied, with particular emphasis to the role of sulfhydryl groups in membrane permeability. The sudden steep rise in the tumor incidence in groups of rats fed 3'-methyl-4-dimethylaminoazobenzene for increasing intervals of time occurs at about 4 weeks. This time correlates with the point of the minimum swelling of microsomes and mitochondria isolated from the livers of rats fed this same dye. Thus, a correlation is established between the alterations of the swelling properties of these particulates and the carcinogenic process.     

Protein carboxyl methyltransferase activity specific for age-modified aspartyl residues in mouse testes and ovaries: Evidence for translation during spermiogenesis

An antiserum prepared against the purified protein carboxyl methltransferase (PCMT) from bovine brain has been used to compare testicular and ovarian levels of the enzyme and to study the regulation of PCMT concentrations during spermatogenesis. The PCMT, which specifically modifies age-damaged aspartyl residues, is present at a significantly higher concentration in mature mouse testis than in ovary. However, the PCMT is present at nearly equal concentrations in extracts of germ cell-deficient ovaries and testes obtained from mutant atrichosislatrichosis mice. In normal testis, the concentration of the PCMT increases severalfold during the first 4–5 weeks after birth, paralleling the appearance and maturation of testicular germ cells. Both immunochemical and enzymatic measurements of PCMT specific activities in purified spermatogenic cell preparations indicate that PCMT levels are twofold and 3.5-fold higher in round spermatids and residual bodies, respectively, than in pachytene spermatocytes. The results are consistent with the enhanced synthesis and/or stability of the PCMT in spermatogenic cells and with the continued translation of the PCMT during the haploid portion of spermatogenesis. The relatively high levels of PCMT in spermatogenic cells may be important for the extensive metabolism of proteins accompanying spermatid condensation or for the repair of damaged proteins in translationally inactive spermatozoa.     

Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization

Cytotactin, an extracellular glycoprotein found in neural and nonneural tissues, influences a variety of cellular phenomena, particularly cell adhesion and cell migration. Northern and Western blot analysis and in situ hybridization were used to determine localization of alternatively spliced forms of cytotactin in neural and nonneural tissues using a probe (CT) that detected all forms of cytotactin mRNA, and one (VbVc) that detected two of the differentially spliced repeats homologous to the type III repeats of fibronectin. In the brain, the levels of mRNA and protein increased from E8 through E15 and then gradually decreased until they were barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridized only to the 7.2-kb message. In isolated cerebella, the 220-kD polypeptide and 7.2-kb mRNA were the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message that contains the VbVc alternatively spliced insert. In situ hybridization showed cytotactin mRNA in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs were more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA was observed in the soma whereas the protein was present externally along the glial fibers. In the telencephalon, cytotactin mRNAs were found in a narrow band at the edge of a larger region in which the protein was wide-spread. Hybridization with the VbVc probe generally overlapped that of the CT probe in the spinal cord and cerebellum, consistent with the results of Northern blot analysis. In contrast, in the outermost tectal layers, differential hybridization was observed with the two probes. In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, was detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes was observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme expressed cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells expressed cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contained cytotactin mRNA. These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissue-specific local signals and prompt a search for functional differences in the various molecular forms of the protein.     

Treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of EGF-stimulated phosphatidylinositol turnover

Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.     

Recent insights in phosphatidylinositol signaling

Studies of phosphatidylinositol signaling pathways are entering a new phase in which molecular genetic techniques are providing powerful tools to dissect the functions of various metabolites and pathways. Studies with phospholipase C are most advanced and clearly indicate that phosphatidylinositol turnover is critical for vision in Drosophila and cell proliferation in various cultured cells. Expression of cDNA constructs and microinjection of PLC or antibodies against it clearly establish a role for PtdIns signaling distinct from its role in calcium mobilization and protein kinase C activation. The importance of inositol cyclic phosphates is also beginning to be realized from the study of cyclic hydrolase using similar techniques. Elucidation of the function of the 3-phosphate inositol phospholipid pathway awaits similar studies. The recent cDNA cloning of inositol monophosphatase (Diehl et al., 1990), Ins(1,4,5)P3 3-kinase (Choi et al., 1990), and inositol polyphosphate 1-phosphatase (York and Majerus, 1991) should provide tools to define further the cell biology of the phosphatidylinositol signaling pathway.     

cDNA cloning and localization of the mouse leukosialin gene (Ly48) to chromosome 7

Mouse leukosialin, previously known as the 3E8 antigen, is expressed primarily on cells of the hematopoietic and lymphoid lineages and is shown to be the mouse homologue to the human leukosialin/sialophorin and rat W3/13 molecules. A partial leukosialin cDNA clone was isolated via cross-species hybridization with a portion of a human leukosialin cDNA. This mouse cDNA clone was used to demonstrate that the leukosialin isoforms are encoded by a single mRNA species of approximately 4.2 kilobases (kb) and that the leukosialin gene is located on chromosome 7. Based on these results, mouse leukosialin is given the designation Ly48.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M30693.