Isolation and partial purification of a new class of transforming growth factors from an avian sarcoma virus-transformed rat cell line.

Transformation of rat cells by avian sarcoma viruses induced the release of growth factors into serum-free conditioned medium. An avian sarcoma virus-transformed rat cell line, 77N1, produced and released a polypeptide growth factor, classified as a transforming growth factor (TGF), which transiently promotes anchorage-dependent BALB3T3 A31 cells to form progressively growing colonies in soft agar. The TGF was isolated and partially purified from an extract of 77N1 cells by ion-exchange chromatography on a diethylaminoethyl Sephacel column followed by ammonium sulfate precipitation. The TGF was assumed to have a molecular weight of 11,000 from gel filtration on Sephadex G-50. This TGF did not compete with epidermal growth factor for binding to cell membrane receptors and was not potentiated by epidermal growth factor. The TGF was trypsin and dithiothreitol sensitive as well as heat and acid labile, indicating that it was different from previously reported TGFs of similar molecular weight and thus belonged to a new class of TGFs.

Slow-Growing Large Irritation Fibroma of the Anterior Hard Palate: A Case Report Using Immunohistochemical Analysis.

Irritation fibromas are recognized as fibrous lesions, usually reactive hyperplasias; however, the mechanism of enlargement is unclear. This paper reports on an abnormally large irritation fibroma of extremely gradual growth. The immunohistochemical features (CD34, α-SMA, vimentin, Ki-67, and TGF-α) of this irritation fibroma are presented to distinguish reactive hyperplasia from other true fibrous neoplasm diseases. In the only previous study, it was reported that the expression of TGF-α might be associated with the development of oral fibromas. Therefore, we investigated the relationship between this exceptionally-large fibrous lesion of extremely slow growth and the immunohistochemical reactivity of TGF-α, finding that, in contrast to the previous study, TGF-α was not expressed. This is the first study to evaluate the enlargement mechanism of such a large irritation fibroma using the approach of immunohistochemical analysis, and it indicates that such analysis can help elucidate the diverse causes and enlargement mechanisms of irritation fibromas.

Effect of transforming growth factor beta on cell proliferation and glycosaminoglycan synthesis by rabbit growth-plate chondrocytes in culture.

The effects of the transforming growth factor beta (TGF-beta) on the growth and glycosaminoglycan synthesis of rabbit growth plate-chondrocytes in culture were studied. In serum-free medium, TGF-beta caused dose-dependent inhibition of DNA synthesis by chondrocytes, measured as [3H]thymidine incorporation (ED50 = 0.1-0.3 ng/ml). The inhibitory effect was maximal at a dose of 1 ng/ml, and extended for a duration of 16-42 h. In contrast, TGF-beta potentiated the synthesis of DNA stimulated by fetal calf serum (FCS). Addition of TGF-beta (1 ng/ml) to cultures containing 10% FCS increased [3H]thymidine incorporation to 1.6-times that in cultures with 10% FCS alone. Consistent with this finding, TGF-beta potentiated DNA synthesis stimulated by the purified growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF). The maximal stimulation of DNA synthesis by FGF (0.4 ng/ml) was further potentiated dose dependently by TGF-beta (ED50 = 0.1 ng/ml, maximum at 1 ng/ml). When the cultures were treated with the optimal concentrations of TGF-beta (1 ng/ml) and FGF (0.4 ng/ml), [3H]thymidine incorporation was 3-times higher than that of cultures treated with FGF alone. This TGF-beta-induced potentiation of DNA synthesis was associated with replication of chondrocytes, as shown by a marked increase in the amount of DNA during treatment of sparse cultures of the cells with the growth factors for 5 days. In contrast, TGF-beta caused dose-dependent stimulation of glycosaminoglycan synthesis in confluent cultures of growth-plate chondrocytes (ED50 = 0.3 ng/ml, maximum at 1 ng/ml). This stimulatory effect of TGF-beta was greater than that of insulin-like growth factor I (IGF-I) or PDGF. Furthermore, TGF-beta stimulated glycosaminoglycan synthesis additively with IGF-I or PDGF. Recently, it has been suggested that bone and articular cartilage are rich sources of TGF-beta, whereas epiphyseal growth cartilage is not. Thus, the present data indicate that TGF-beta may be important in bone formation by modulating growth and phenotypic expression of chondrocytes in the growth plate, possibly via a paracrine mechanism.

Inhibition of different steps of the ubiquitin system by cisplatin and aclarubicin.

Ubiquitin is involved in such fundamental cellular processes as cell cycle control, DNA repair, protein degradation and stress responses. We previously reported that cisplatin could inhibit the ubiquitin-ATP-dependent proteolysis and ubiquitination. We further investigated the effect of various antitumor agents on the ubiquitin system and found that aclarubicin (ACR) inhibits the ubiquitin-ATP-dependent proteolysis but not the ubiquitination process. We found that ACR as well as cisplatin inhibited the ubiquitin-ATP-dependent proteolytic activity of rabbit reticulocytes. The IC50 values of these agents were 52 and 90 microM, respectively. Although cisplatin inhibits the conjugation of ubiquitin to proteins through the inhibition of a ubiquitin-activating enzyme, ACR, at 120 microM, does not. Thus, the antitumor agents affecting the ubiquitin system could be classified into two groups; one is represented by cisplatin, which inhibits the ubiquitination of the proteins, and the other is ACR, which does not inhibit the ubiquitination but does inhibit the ubiquitin-ATP-dependent proteolysis. Mitomycin C belongs to the latter group.

Reversal of neuronal polarity characterized by conversion of dendrites into axons in neonatal rat cortical neurons in vitro.

The mechanisms for the establishment and maintenance of cell polarity in neurons are not well understood. Axon regeneration from dendrites has been reported after axotomy near the cell body in vivo. We report here in vitro a reversal of neuronal polarity characterized by the conversion of dendrites into axons. We isolated neurons from the neonatal rat cerebral cortex. Neurons that exhibited an apical dendrite with a length of >100 microm were monitored for 3 days in culture. In 66% of neurons examined, a new axon, as identified by reactivity with an antibody to dephosphorylated tau or by lack of reactivity with an antibody to the a and b isoforms of microtubule-associated protein 2, appeared to form from the tip of the original dendrite. Further analysis of such neurons revealed that the distal half of the original dendrite became positive for dephosphorylated tau or negative for microtubule-associated protein 2. Time-lapse video microscopy demonstrated the conversion of the original dendrite into an axon without dendritic retraction. Axon regeneration from dendritic tips required a significantly longer time than axon regeneration from minor processes. Our observations thus demonstrate in vitro a time-consuming reversal of neuronal polarity and the conversion of a dendritic cytoskeleton into an axonal one.

Microsatellite instability correlates with normal expression of cyclin E in hepatocellular carcinomas.

It has been reported that microsatellite instability (MSI) strongly correlates with carcinogenesis and cancer progression. In the present study, we studied the incidence of MSI at 5 polymorphic microsatellite markers (D5S406, D13S153, D16S402, D17S796, and poly(A) tract BAT26), the expression of G1 cyclins (cyclin A, cyclin D and cyclin E), and Ki-67 labeling index in 30 surgically resected hepatocellular carcinomas (HCCs) and their adjacent non-cancerous tissues. The results of analysis showed that 43% of HCCs exhibited MSI in one locus, 10% in two loci, and 3% in three loci. Overexpressions of cyclin E and cyclin A were observed in 57% and 83% of HCCs, respectively. MSI in HCCs, however, correlated with normal expressions of cyclin E and cyclin A and with a low labeling index of Ki-67. Thus, patients with HCCs exhibiting MSI at these 5 markers may have less involvement of G1/S disregulation and may have better prognosis than other patients with HCC.

Different expression of positive and negative regulators of hepatocyte growth in growing and shrinking hepatic lobes after portal vein branch ligation in rats.

Portal vein branch embolization is often performed before hepatectomy to prevent postoperative liver failure. It is, however, still not clear how the embolized lobe shrinks and the non-embolized lobe proliferates in counterbalance. We investigated the expression of positive and negative regulators of hepatocyte growth to clarify the mechanisms of liver growth and atrophy in a rat portal vein ligation (PVL) model compared with partial hepatectomy (PH). A significant increase in DNA synthesis within the non-ligated lobe reached a peak at 36 h, a delay of 12 h as compared with PH, while no increase occurred in the ligated lobe. Expression of hepatocyte growth factor mRNA remarkably increased in the non-ligated growing lobe between 6 and 24 h, but was only slightly elevated in the ligated shrinking lobe. Contrarily, negative regulators of hepatocyte proliferation, such as TGF-beta1 and IL-1beta, were strongly expressed in the ligated shrinking lobe. Thus, the changes of portal venous flow and/or pressure caused by PVL may contribute to induction of different kinds of growth factors between the ischemic and non-ischemic lobes; these factors possibly regulate liver regeneration and atrophy after PVL.

Inhibition of ubiquitin-ATP-dependent proteolysis and ubiquitination by cisplatin.

We tested the inhibitory activity of various antitumor agents on the ubiquitin-ATP-dependent proteolytic activity in rabbit reticulocyte lysates. We found that cisplatin, 4'-(9-acridinyl-amino) methanesulfon-m-anisidide (m-AMSA) and mitomycin C inhibited the ubiquitin-ATP-dependent proteolysis. IC50 values (50% inhibition concentrations) of these antitumor agents were 90, 210 and above 290 microM, respectively. Furthermore, cisplatin was found to inhibit the conjugation of ubiquitin to endogenous proteins in fraction II at 100 and 330 microM. These results suggest that cisplatin interacts with the enzyme(s) involved in ubiquitin conjugation and thus inhibits the ubiquitin-ATP-dependent protein degradation. We assume that the agents that can affect the ubiquitin system might be useful for the treatment of tumors and that the ubiquitin system could be a new target for cancer chemotherapy.

Enhanced expression of cyclin E and cyclin A in human hepatocellular carcinomas.

BACKGROUND: Disruption of the G1/S check point leads to uncontrolled cell growth, resulting in the development of cancers. MATERIALS AND METHODS: Cell cycle regulatory molecules were investigated in 33 surgically resected hepatocellular carcinoma (HCC) samples from 30 patients by Western blotting. RESULTS: Enhanced expressions of cyclin E and cyclin A were detected at frequencies of 18/33 and 26/33 in HCCs, respectively, as compared with their neighboring noncancerous tissues. The enhanced expression of cyclin E, but not that of cyclin A, correlated with hyperphosphorylation of pRb and high frequency of Ki-67-positive cells. Thus, the HCCs with enhanced cyclin E expression probably contain a relatively large number of proliferating cancer cells. CONCLUSIONS: The degree of cyclin E expression can be used as a prognostic parameter of HCC. In addition, cyclin E may become a molecular target in the treatment of HCCs.

Establishment of a human hepatoma cell line, HLE/2E1, suitable for detection of p450 2E1-related cytotoxicity.

By transfection of an expression vector of human cytochrome P450 2E1 (CYP2E1) into a human hepatoma cell line (HLE), a new cell line (HLE/2E1) that stably expresses activity of CYP2E1 has been established. The HLE/2E1 cell line expressed a higher level of CYP2E1 messenger ribonucleic acid than did the mother HLE cell line. CYP2E1 enzyme activity determined by a p-nitrophenol oxidation assay was also higher in HLE/2E1 cells than in HLE cells. In addition, the enzyme activity of the HLE/2E1 cells was increased by ethanol treatment. Exposure to acetaminophen (APAP) or buthionine sulfoximine (BSO) caused a greater decrease in viability of the HLE/2E1 cells than that of the HLE cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The cytotoxicity of APAP or BSO to HLE/2EI cells was inhibited by the addition of ethanol or vitamin E. However, the cytotoxicity of both APAP and BSO was enhanced by 24-h preincubation of HLE/2E1 cells with ethanol. These results show that this cell line provides a useful model for studying catalytic properties of CYP2E1 and cytotoxic mechanisms of chemicals metabolized by CYP2E1.