The structurally diverse xenobiotic peroxisome proliferators (PPs) increase the number of peroxisomes per cell and the levels of several enzymes, and cause hepatomegaly, often leading to hepatocarcinogenesis in a species- and tissue-specific manner. The deadlocked problems of the molecular mechanism of PP action and its physiological meanings have begun to be understood through cDNA cloning of a PP-activated receptor (PPAR). PPAR, a member of the steroid/thyroid/vitamin superfamily of nuclear receptors, has isoforms and differentially heterodimerizes with other nuclear receptors, providing potential mechanisms not only for species-and tissue-specific actions but also for diverse actions of PPs. Recent findings related to PPAR are summarized, and its possible role in lipid metabolism and involvement in PP-induced hepatocarcinogenesis are discussed.

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The presence of body-like structures in nuclei from interferon alpha-treated Daudi cells has been shown on the ultrastructural level, by the use of different staining methods. The degree of their rearrangement in the nucleoplasm seems to be dependent on the time of interferon treatment. Since this morphological EVIDENCE has been found to be preceded by a slowing down of RNA transcriptional machinery early upon the interferon administration, it is speculated that interferon generated signals might lead to RNP granule accumulation in the nucleus and a consequent arrangement into defined structures.

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Quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV) proliferate and do not differentiate at 35.5°C, the permissive temperature for the virus, whereas their myoblast differentiation proceeds at 41.0°C, a non-permissive temperature. In this experimental system, myogenic differentiation is controlled by src gene products. Using QM-RSV cells as an antigen, a monoclonal antibody, Mb-N3, was prepared. Expression of Mb-N3 antigen was found to increase during differentiation. Therefore, in studies on the mechanism of myogenic differentiation, we examined the expression of Mb-N3 antigen on spontaneously forming myotubes formed at 41.0°C and fused myoblasts with hemagglutinating virus of Japan (HVJ, Sendai virus) disregarding programmed processes for myogenic differentiation.
When the myoblasts cultured at 35.5°C were treated with HVJ, they fused with each other. These fused myoblasts were elongated and were morphologically similar to spontaneously forming myotubes. Thus, we called fused myoblasts with HVJ "artificial myotubes. " During culture at 35.5°C, the artificial myotubes did not show increased expression of Mb-N3 antigen and increase of creatine kinase activit, which are markers of normal biochemical differentiation. When artificial myotubes were cultured at 41.0°C, expression of Mb-N3 antigen and creatine kinase activity increased. These results suggest that the expression of the antigen is regulated by kinase activity derived from src gene products even after compulsory cell fusion. Moreover, compulsory fusion does not cause myogenic differentiation and expression of Mb-N3 antigen. Thus it seems that the differentiation program must proceed in order for myogenic differentiation and expression of Mb-N3 antigen to take place.

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The expression of J chain and μ chain genes has been investigated during human fetal ontogeny by the polymerase chain reaction performed on liver sections. With this technique, we have been able to detect expression of J chain and μ chain in a single 6-10-μm-thick formalin-fixed paraffin-embedded section of fetal liver tissue. J chain expression reached a maximum at 16 weeks of gestation but was clearly detectable in the liver at the 6th week. Although not detectable at the 6th week, μchain mRNA became readily detectable at 7 weeks of gestation. These results indicate that J chain expression precedes that of μchain by at least one week. Therefore, our results imply that J chain is the first immunoglobulin-related polypeptide expressed during the embryogenesis and differentiation of B cells in the fetal liver.

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The effects of various growth factors on the colony-forming ability were examined in soft-agar culture of thermosensitive (ts) mutants derived from Chinese hamster lung cells that exhibited anchorage-independent growth. Fibroblast growth factor (FGF), a competence factor, and epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), progression factors, did not stimulate colony formation in soft-agar culture of any of the ts cells. Insulin stimulated colony formation in soft-agar culture of some of the thermosensitive mutants at the nonpermissive temperature. These results suggested that the interruption of growth occurred late in the Gi phase of the cell cycle in these mutant cells. Mutants were classified into two groups: members of Group 1 could be stimulated by insulin but not by FGF, EGF or IGF-I (5 clones); members of Group 2 were not stimulated either by insulin, FGF, and EGF or by IGF-I (4 clones). Insulin-binding capacities of mutants designated cs-17-25 and hs-211 did not differ between cells cultured at a nonpermissive and a permissive temperature. The extent of phosphorylation of the insulin receptors of these clones changed depending upon the dose of insulin. However, there were no differences in the extent of autophosphorylation of receptors between the mutant cells cultured at the permissive and the nonpermissive temperature. Therefore, thermosensitivity of colony-forming ability is not the result of any decrease in insulin-binding capacity or in activity of the receptor kinase. These results suggest that the thermosensitive lesions of these ts mutants occur distal to the transduction of a growth signal that is mediated by the kinase activity of the insulin receptor.

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Normal human mammary epithelial cells (HMEC) isolated from surgically resected breast tissues were cultured under serum-free conditions using MCDB170 medium. With the increase in the number of passages, in particular after the 5th passage, the number of enlarged, flattened and vacuolated cells increased while cell proliferation decreased. The senescent cells occasionally had keratohyaline granules in the cytoplasm and were positive in immunohistochemistry for keratinizing squamous epithelium-specificcytokeratin 10. When HMEC were cultured between floating double-layered collagen gels, the cells lost growth activity, showed marked stratification, and became positive for carcinoembryonic antigen (CEA). The stratified cells underwent
squamous differentiation and tonofilament bundles appeared around the nuclei. The stratification and squamous differentiation of HMEC were observed within seven days after transfer to the three-dimensional culture, regardless of the number of passages. These results indicate that the HMECin vitro ultimately differentiate into squamous epithelia and also that there is a close relationship between the squamous-type differentiation and the loss of cell proliferation.

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Physarum vitronectin-like protein with a molecular mass of 70 kDa cross-reacts with antibovine vitronectin and promotes cell-spreading (MIYAZAKI, K. et al. 1992. Exp. Cell Res., 199: 106-110.). The amino-terminal sequence of Physarum vitronectin-like protein is, however, distinct from those of animal vitronectins but shows significant sequence homology with dihydrolipoamide acetyltransferase, a component of pyruvate dehydrogenase complex. We have investigated the structural relationships between Physarum vitronectinlike protein and dihydrolipoamide acetyltransferase by using both antibody and protein-chemical methods. The vitronectin-like protein reacted with both anti-bovine vitronectin IgG and anti-rat pyruvate dehydrogenase complex IgG, indicating that it shares common antigenic determinant(s) with rat pyruvate dehydrogenase complex. Furthermore, sequencing studies of peptides obtained by lysylendopeptidase digestion indicated that internal sequences of Physarum vitronectin-like protein show significant homology with dihydrolipoamide acetyltransferase, but do not show any homology with the primary structures of authentic vitronectins. Immunocytochemistry revealed that the protein is widely localized in cytoplasm and nuclei of Physarum polycephalum, but is not present in the central area of vacuoles. Our results indicate that Physarum vitronectin-like protein is a molecule structurally and immunologically related to dihydrolipoamide acetyltransferase but functionally similar to animal vitronectin, although its localization is unique.

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The localization of basic fibroblast growth factor (bFGF) in various cultured bFGF-producing cells, including bovine capillary endothelial (BCE) cells and human cholangiocellular carcinoma (HuCC-Tl) cells, was determined by measuring binding of ¹²⁵I-labeled specific monoclonal antibody against bFGF. bFGF on the cell surface and within the cells was determined by counting the radioactivity of the labeled monoclonal antibody bound to the cells before and after increasing their permeability by treatment with alcohol or Triton X-100. The radioactivity bound to these cells decreased with increase in the concentration of unlabeled monoclonal antibody. Scatchard analyses of these data suggested the quantitative localization of bFGF and Kd values showing the specific binding. This method was designated as in situ assay with radio-labeled antibody (ISARA). bFGF detected on the cell surface and within the cells could be partially removed by treatment with heparin or heparinase and with heparin or DNase, respectively. Exogenous bFGF bound to cells not producing bFGF was also quantified by ISARA. Measurements of bFGF in extracts of cells producing bFGF suggested that 8.3-13.3% of the bFGF associated with the cells was detected by ISARA. This method should be useful for quantitative confirmation of immunohistochemical results on bFGF.

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Epithelial-like cells from the colorectum of one-day-old newborn rats were immortalized by transfection with the simian virus 40 (SV40) T-antigen gene, and a cell line OUMS-25 was established. The cells were positive for the SV40 T-antigen, and immunoreactive to a colonic epithelial cell monoclonal antibody and a keratin-18 monoclonal antibody. Ultrastructural studies revealed the presence of microvilli on the cell surface and desmosomes between the adjacent cells. Karyotypic analysis showed that OUMS-25 cells were aneuploid. Cloning efficiency of the cells was 0.01% in soft agar. However, the cells were not tumorigenic in the syngeneic newborn rats. The cells were further transformed by transfection with the cloned activated c-Ha-ras oncogene containing a point mutation within codon 61. Characteristics of the activated-c-Ha-ras transfected cells (OUMS-25/RAS) were different in some respects from those of the parent cells (OUMS-25). OUMS-25/RAS cells demonstrated more malignant morphology, elevated cloning efficiency in soft agar, and tumorigenicity. This is the first report on the immortalization and malignant transformation of colorectal epithelial-like cells by transfection with a combination of SV40 T-antigen gene and cloned activated c-Ha-ras- oncogene.

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The production of proteolytic emzymes by osteoblasts is considered important for initiating osteoclastic bone resorption. Using the established cell line NY as an example of osteoblast-like cells, the effect of intracellular cyclic AMP (cAMP) and protein kinase C (PKC) on plasminogen activator secretion and its specific binding to the cells were investigated. HT-1080 cells were used as the control. NY cells predominantly secrete single-chain urokinase-type plasminogen activator (scu-PA) and some two-chain u-PA. Both scu-PA and u-PA were present in the cell surface and cell lysate of NY cells, and their distribution in HT-1080 cells was quite similar to that of NY cells. Exposing cells to phorboi myristate acetate (PMA) or dibutyryl cyclic AMP (db cAMP) enhanced the secretion of scu-PA and two-chain u-PA, whereas 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) decreased scu-PA secretion, indicating that it is enhanced by protein kinase C (PKC) as well as by cAMP in NY cells. On the other hand, in HT-1080 cells, PMA decreased the level of two-chain u-PA secretion into the conditioned medium. The binding assay of ¹²⁵I-DFP-u-PA to NY cells revealed the presence of a single class of binding sites with a K_d of 2.23 nM and B_max of 0.82×10⁶ binding sites/cell. PMA however, altered neither the K_d nor the B_max. Dibutyryl cAMP increased the B_max 1.9 fold. Thus, NY cells secrete u-PA and express specific binding sites on the cell surface, which are modulated by cAMP and PKC. The u-PA/u-PA receptor system may contribute to osteoblastic bone resorption.

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The unique Physarum lysophosphatidic acid, PHYLPA, having a cyclopropane in the fatty acid moiety and a cyclic phosphate at C-2 and C-3 positions of the glycerol (16), inhibited proliferation of human fibroblast cells, TIG-3 and TIG-7, which were cultured in a chemically defined (serum-free) medium. The cells at S- and M-phases proceeded to G₂- and G₁-phases, respectively, and most of cells were arrested at G₁- or G₂- phase during PHYLPA treatment. The growth was recovered when PHYLPA was removed from the medium. In the presence of serum, PHYLPA did not show obvious inhibitory effects, indicating the existence of a factor(s) which neutralizes the antiproliferative activity of PHYLPA.
PHYLPA elicited an increase in 3', 5' -cyclic adenosine monophosphate (cAMP) in a biphasic fashion in fibroblast cells. It also elicited inositol phosphate accumulation, as well as a transient rise in cytoplasmic free Ca²⁺ ion.

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