Effects of hexamethylene bisacetamide onα-fetoprotein,albumin, and transferrin production by two rat hepatoma cell lines

Summary α-Fetoprotein (AFP), albumin, and transferrin production by two rat hepatoma cell lines, McA-RH 7777 (7777) and McA-RH 8994 (8994), was determined after treatment with hexamethylene bisacetamide (HMBA, 2 to 6 mM). Radioimmunoassays were used to determine the levels of both secreted and intracellular AFP, albumin, and transferrin. Line 7777 normally produces large quantities of AFP and small quantities of albumin, thus resembling the less differentiated fetal liver with respect to the synthesis of these two proteins. Line 8994 normally produces small quantities of AFP and relatively larger amounts of albumin, thus resembling hepatic functions characteristic of a more differentiated state. After treatment with HMBA for a period of 28 to 96 h a threefold increase in AFP secretion by 7777 and a dose related increase in AFP, albumin, and transferrin secretion by 8994 were observed. In contrast, the secretion of albumin and transferrin in 7777 was inhibited by 60 and 40%, respectively, following treatment with HMBA. The intracellular concentrations of AFP in 7777 and AFP, albumin, and transferrin in 8994 were increased by treatment with HMBA indicating that HMBA is able to stimulate the synthesis of these proteins. The intracellular concentration of AFP, albumin, and transferrin in 7777, when expressed as a percentage of the extracellular concentration of these proteins, did not change significantly during HMBA treatment, indicating that the observed decrease in secreted albumin and transferrin by 7777 is due to decreased synthesis. Similarly, in Line 8994, when the intracellular concentration of the three proteins was expressed as percentage of the extracellular concentration, the only significant change observed was an increase in AFP after 72 h of HMBA (5 mM) treatment. The observed changes in the synthesis of AFP, albumin, and transferrin in both 7777 and 8994 after HMBA treatment were reversible, as judged by the return to control values upon removal of HMBA from the culture medium. Thus, HMBA stimulates synthesis of the oncofetal protein AFP, a result that appears to be independent of the stage of differentiation of the cell. However, its effect on the synthesis of albumin and transferrin are opposite in the two cell lines, suggesting that the regulation of the synthesis of these two proteins is controlled by factors or conditions that are dependent upon the stage of differentiation of the hepatoma cell lines.     

Differential effects of dimethyl sulfoxide and sodium butyrate on alpha-fetoprotein, albumin, and transferrin production by rat hepatomas in culture

Radioimmunoassay was used to determine alpha-fetoprotein (AFP), albumin, and transferrin production (ng/10(5) cells/24 h) by two cell lines (7777 and 8994) derived from chemically induced rat hepatomas. alpha-Fetoprotein production was high (2000 to 4400) in 7777, but was very low (0.2 to 0.4) in 8994. Albumin production varied from 0.4-0.8 (7777) to 14-26 (8994). Both lines produced substantial amounts of transferrin (180 to 240 by 7777 and 29 to 42 by 8994). Addition of dimethyl sulfoxide (DMSO, 1 to 4%) or sodium butyrate (BA, 0.5 to 2.0 mM) to the medium inhibited growth in both lines, but 8994 was more sensitive to these agents than 7777. Dimethyl sulfoxide treatment (2 to 4%) resulted in a dose-related decrease (less than 10% of control at 4% DMSO) in AFP, albumin, and transferrin production by 7777, but in 8994, DMSO (1 to 2%) resulted in an increase (up to sixfold) in albumin and transferrin production, without affecting AFP production. By contrast, BA (2 to 4 mM) stimulated the production of all three proteins in both lines, most notably that of albumin (up to sixfold) by 7777 and that of AFP (up to 20-fold) by 8994. It is concluded that both DMSO and BA can enhance the expression of differentiated functions of the hepatoma cell, and that DMSO at the same time can suppress the expression of an oncofetal function. However, neither DMSO nor BA is selective in its effects on specific genes (i.e., normal, adult vs. oncofetal genes), and it appears that their effects may be the result of a more general phenomenon, the expression of which may be related to the stage of differentiation of the cell.     

Differential regulation of tissue transglutaminase in rat hepatoma cell lines McA-RH7777 and McA-RH8994: Relation to growth rate and cell death

Close correlation between tissue transglutaminase (tTG) induction and growth regulation and/or cell death processes has been suggested in many cell lineages. In this study, the regulation of the tTG levels by various growth and differentiation factors and its relation to growth rate and cell death processes were investigated in two rat hepatoma cell lines, McA-RH7777 and McA-RH8994, using a monoclonal antibody against liver tTG. Transforming growth factor-β1 (TGF-β1) and retinoic acid (RA) each increased tTG to the level of 8- to 32-fold above that of control cultures in both cell lines after 72-h treatment. Dexamethasone (DEX) induced a 16- to 32-fold of tTG in McA-RH8994 cells while it did not change the enzyme level in McA-RH7777 cells. Simultaneous addition of DEX and RA increased the tTG level to more than 50-fold in McA-RH7777 cells as well as McA-RH8994 cells. Other factors, such as TGF-α, hepatocyte growth factor, dimethyl sulfoxide, and protein kinase C activator, did not show significant increases of the tTG levels. Although tTG induction by TGF-β1 or DEX appeared to be correlated with their growth suppressive effects, RA increased the tTG level without suppressing the growth rate of hepatoma cells. TGF-β1 was also shown to induce cell death in both cell lines. Our results demonstrate that RA and DEX are capable of modulating the TGF-β1-induced cell death processes independent of the tTG levels. We present evidence here that tTG induction by itself is not the direct cause of growth suppression and cell death in these hepatoma cells.     

Differential effects of dimethyl sulfoxide and sodium butyrate on α-fetoprotein,albumin, and transferrin production by rat hepatomas in culture

Summary Radioimmunoassay was used to determine α-fetoprotein (AFP), albumin, and transferrin production (ng/10⁵ cells/24 h) by two cell lines (7777 and 8994) derived from chemically induced rat hepatomas. α-Fetoprotein production was high (2000 to 4400) in 7777, but was very low (0.2 to 0.4) in 8994. Albumin production varied from 0.4–0.8 (7777) to 14–26 (8994). Both lines produced substantial amounts of transferrin (180 to 240 by 7777 and 29 to 42 by 8994). Addition of dimethyl sulfoxide (DMSO, 1 to 4%) or sodium butyrate (BA, 0.5 to 2.0 mM) to the medium inhibited growth in both lines, but 8994 was more sensitive to these agents than 7777. Dimethyl sulfoxide treatment (2 to 4%) resulted in a dose-related decrease (<10% of control at 4% DMSO) in AFP, albumin, and transferrin production by 7777, but in 8994, DMSO (1 to 2%) resulted in an increase, (up to sixfold) in albumin and transferrin production, without affecting AFP production. By contrast, BA (2 to 4 mM) stimulated the production of all three proteins in both lines, most notably that of albumin (up to sixfold) by 7777 and that of AFP (up to 20-fold) by 8994. It is concluded that both DMSO and BA can enhance the expression of differentiated functions of the hepatoma cell, and that DMSO at the same time can suppress the expression of an oncofetal function. However, neither DMSO nor BA is selective in its effects on specific genes (i.e., normal, adult vs. oncofetal genes), and it appears that their effects may be the result of a more general phenomenon, the expression of which may be related to the stage of differentiation of the cell.     

Bidirectional morphological changes induced by dexamethasone in Morris hepatoma cell lines McA-RH7777 and McA-RH8994: independence of fibronectin and its receptor.

Two Morris hepatoma-derived cell lines, McA-RH7777 (7777) and McA-RH8994 (8994), exhibit different alterations in morphology upon exposure to glucocorticoid. After treatment with synthetic glucocorticoid dexamethasone (DEX), 7777 cells show increased adhesiveness and more flattened shape, while DEX-treated 8994 cells show decreased adhesiveness to substratum and exhibit a marked increase of round and detached cells. Since fibronectin has been thought to play an important role in cell adhesiveness to substratum in hepatoma cell culture, we have also compared the effects of DEX on the biosynthesis of fibronectin (FN) and the functional level of FN receptor in 7777 and 8994 cells. Northern blot analysis and immunofluorescent studies showed that 7777 cells have a high basal expression level of FN synthesis and that DEX treatment induces FN expression two- to threefold with establishment of an extensive fibrillar FN network around the cells. On the other hand, 8994 cells were shown to express little FN and no apparent FN was localized on nonstimulated 8994 cells. However, DEX-treatment drastically increased FN expression in 8994 cells to the level of more than that of DEX-treated 7777 cells and induced a detectable level of cell-associated FN around DEX-treated 8994 cells, which appears to be contradictory to the decreased adhesiveness to the substratum in DEX-treated 8994 cells. Cell attachment assays using FN-coated plates demonstrated that DEX does not exhibit significant effects on the attachment of either 7777 or 8994 cells to FN-coated dishes. Our results suggest that decrease of adhesiveness to the substratum and increase of round detached cells in DEX-treated 8994 cells are independent of changes in the FN expression and the function of FN receptor.     

丁酸钠至少部分通过NF-Y 依赖的TXNIP 表达诱导人肺癌细胞A549死亡

组蛋白去乙酰化酶（HDACs）抑制剂丁酸钠调节细胞分化、增殖和抑制肿瘤发生。硫氧还蛋白相互作用蛋白( thioredoxin-interacting protein,TXNIP)通过负性调控硫氧还蛋白的活性,调控细胞内的氧化还原平衡,抑制细胞生长。本研究证明,丁酸钠可通过激活依赖于转录因子NF-Y的TXNIP 表达,诱导人非小细胞肺癌细胞A549死亡。MTT法显示,5 mmol/L丁酸钠处理A549 细胞72 h可显著诱导其死亡;流式细胞分析发现,其中大部分细胞以凋亡形式死亡。表达芯片分析表明,在丁酸钠处理的A549 细胞中,TXNIP 的mRNA 水平显著提高30～50倍;实时定量PCR、免疫细胞化学和蛋白质印迹结果进一步证明,丁酸钠可显著上调TXNIP 表达。荧光素酶报告基因分析证明,与对照细胞比较,丁酸钠刺激的细胞内报告酶活性可提高约10 倍,提示丁酸钠可激活TXNIP 启动子的转录活性。TXNIP 启动子删除突变分析显示,删除NF-Y 结合的DNA 序列显著降低丁酸钠对TXNIP 启动子的激活能力, 表明NF-Y转录因子参与丁酸钠介导的TXNIP基因转录激活。为分析TXNIP 在A549 细胞中的定位和部分功能,在A549细胞 中过表达GFP TXNIP 融合蛋白及其截短突变体融合蛋白;结果显示,野生型和保留N 端1-281aa的截短突变体定位在细胞核,而删除N 端1-200aa 时,其定位在细胞核和细胞质,提示N 端1 200aa 可调节该蛋白质的定位。然而,丁酸钠刺激未发现表达的GFP TXNIP在细胞内定位改变。以上结果表明,丁酸钠可通过激活转录因子NF YC 依赖的TXNIP激活,诱导A549 细胞死亡,但不能改变TXNIP蛋白在细胞内的定位。上述结果还提示,TXNIP 的N 端1-200aa 可能在调节TXNIP 的细胞定位中发挥作用。是否丁酸钠刺激TXNIP表达导致的细胞死亡系通过改变细胞氧化压力,以及TXNIP在细胞中定位的详尽调节机制尚待进一步研究证明。     

Modulation of the expression of a multidrug resistance gene (mdr-1/P-glycoprotein) by differentiating agents

Acquired resistance to multiple natural products in vitro is mediated by P-glycoprotein (Pgp). Expression of this protein has been demonstrated in some normal tissues and in tumor samples obtained from both untreated and treated patients. In situ hybridizations with RNA probes have demonstrated higher levels of expression of mdr-1/Pgp in well-differentiated tumors and in well-differentiated areas in tumors with mixed histologies. Expression of mdr-1/Pgp in human colon carcinoma cell lines was increased by the differentiating agents sodium butyrate, dimethyl sulfoxide, and dimethylformamide. In the SW-620 cell line addition of sodium butyrate resulted in a rapid induction of mdr-1/Pgp mRNA that was sustained for the duration of the exposure. The levels of P-glycoprotein were measured by immunoblotting and were also increased. Similar results were obtained in three other cell lines including the HCT-15 line. This induction occurred without alterations in nuclease sensitivity. Discontinuation of sodium butyrate was followed by a rapid fall in the levels of mRNA. The levels of P-glycoprotein returned to normal with a half-life of about 24 h. In spite of a 20-25-fold increase in the level of mdr-1/Pgp mRNA and P-glycoprotein, the SW-620 cell line did not demonstrate increased resistance to doxorubicin and vinblastine or decreased accumulation of vinblastine. In contrast, in the HCT-15 cell line, a 5-fold increase of mdr-1/Pgp was accompanied by a comparable fall in vinblastine accumulation which was reversed by verapamil. In the SW-620 cell line, the induced protein could be photolabeled using [3H]azidopine. Expression of mdr-1/Pgp appears to correlate with the degree of differentiation. However, its induction is not always accompanied by expression of the multidrug-resistance phenotype.     

Butyrate effects on normal and adapted hepatoma cells: morphological response and implications for vectoral cholesterol transport

The cell line 4IC6, adapted for growth in 6 mM sodium butyrate from Hepatoma Tissue Culture cells [R. Chalkley, and A. Shires (1985) J. Biol. Chem. 260, 7698-7704], exhibits a fourfold increase in histone acetate turnover. The 4IC6 cells were about 25 times more resistant to butyrate relative to the parental cell line as measured by cloning efficiency. This line also maintains a flatter and more extended morphology when growing in the presence of 6 mM sodium butyrate relative to the parental line. Both cell lines maintain similar intracellular butyrate levels and incorporate [1-14C]butyrate into lipids to similar extents when incubated in medium containing high levels of the fatty acid. These results show that 4IC6 cells have not attained butyrate resistance through acquiring the ability to metabolize butyrate more efficiently or in a significantly different manner when compared with the parental cell line. The membrane lipid composition was nearly identical between the two cell types. Thus the different morphologies exhibited by each cell line were not a consequence of altered membrane lipid composition. The resistant line, 4IC6, maintains about 10-fold higher cholesterol ester levels and half the level of triglycerides found in the parental line. The butyrate-resistant cells also synthesize cholesterol at about a 1.8-fold higher rate than do the parental cells. This difference in de novo synthesis is reflected by a difference of a similar factor in the amount of radioactive cholesterol the two cell lines accumulate over 12 generations. These results are discussed with respect to models for equilibration of serum lipoprotein-derived and newly synthesized cholesterol.     

DISRUPTION OF CELL—CELL ADHESION IN THE PRESENCE OF SODIUM BUTYRATE ACTIVATES EXPRESSION OF THE 92kDa TYPE IV COLLAGENASE IN MDCK CELLS

We have investigated the effects of altered cell shape on the regulation of the 92kDa type IV collagenase. In MDCK cells, anti-E-cadherin antibodies alter cell shape by disrupting normal cell—cell contacts, while sodium butyrate causes a marked flattening and spreading of cells. The disruption of cell—cell contacts led to a faint expression of the 92kDa collagenase. This effect was enhanced by sodium butyrate, which by itself did not induce collagenase expression. In contrast, stromelysin expression was not regulated in these conditions. Although mRNA expression was enhanced, the secreted collagenase activity was not altered in these conditions in either cell line. Examination of cytoskeletal and extracellular matrix proteins and cell—cell and cell—matrix adhesion proteins by immunofluorescence and Western blot revealed a disruption of the actin network, tight junctions, and fibronectin deposition by anti E-cadherin antibodies, and alterations in actin, cytokeratin 8, cytokeratin 14, laminin and β1 integrin induced by sodium butyrate. Thus, the induction of collagenase expression in epithelial cells by disrupted cell—cell adhesion and sodium butyrate is associated with changes in cell shape and structure.