Down-regulation of statin,a nonproliferation-specific nuclear protein,and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G₁ phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G₁ phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley-Liss, Inc.     

Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells

Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-kappaB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-x(L) and cyclin D2, regulated by NF-kappaB. Although the viral protein Tax is an activator of NF-kappaB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-kappaB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.     

c-myc-dependent hepatoma cell apoptosis results from oxidative stress and not a deficiency of growth factors

Expression of c-myc regulates apoptotic cell death in the human hepatoma cell line HuH-7 during culture in serum-free medium (SFM) plus zinc. To understand the mechanism of this c-myc effect, the ability of various serum-contained factors to prevent apoptosis was determined. Apoptosis was not inhibited by growth factors and was even accelerated by supplementation with insulin-like growth factor I or insulin. Cell death was prevented by SFM supplementation with the amino acid glutamine but not serine or asparagine. Improved cell survival with glutamine was associated with increased levels of glutathione (GSH). In HuH-7 cells cultured in SFM plus zinc, c-myc expression led to decreased levels of GSH, and elevated intracellular levels of hydrogen peroxide (H₂O₂). Cell death induced by c-myc expression was inhibited by the addition of catalase or dimethyl sulfoxide, a hydroxyl radical scavenger, or by increased intracellular expression of catalase. In contrast to findings in fibroblasts, c-myc-dependent apoptosis during serum deprivation in HuH-7 hepatoma cells was unrelated to a loss of growth factors. Apoptosis resulted from H₂O₂-mediated oxidative stress with associated glutamine dependent intracellular GSH depletion. J. Cell. Physiol. 170:192–199, 1997. © 1997 Wiley-Liss, Inc.     

Cell cycle-dependent gene expression in V point-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G₁ at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G₁ and enter S phase. As data presented here show that mRNA synthesis is needed for 2–3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid-G₁ arrest protocol to analyze gene expression in late G₁. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G₁, genes normally expressed in late G₁ were expressed only after release from the V point. The expression of late G₁ genes in cells released from the V point was temporally similar, in respect to G₁ location, as was seen in stimulation of quiescent G_o cells. As this protocol effectively divides gene expression into early (pre-V point) and late (post-V point) categories, it should be useful in studies of growth factor-modulated events that regulate traverse of late G₁ and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G₁, is required for BALB/c-3T3 fibroblasts to traverse late G₁ and initiate DNA synthesis. © 1993 Wiley-Liss, Inc.     

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-myc and resultant depression of proliferation. © 1996 Wiley-Liss, Inc.     

Suppression of tumorigenicity in T-cell lymphoma hybrids is correlated with changes in myc expression and DNA replication of the myc chromosomal domain

Intraspecific somatic cell hybrids between T-lymphoma cells and lymphocytes are highly tumorigenic whereas fusion of T-lymphoma cells with normal fibroblasts leads to reduced or even completely suppressed tumorigenicity of the hybrid cells. A particular cytogenetic phenomenon defines these two classes of hybrids. DNA replication analysis via bromodeoxyuridine pulse labelling reveals an aberrant banding pattern in the c-myc chromosomal domain in tumour cells and highly tumorigenic hybrids. In hybrids with suppressed tumorigenicity the tumour parent derived chromosomes have reverted to normal DNA replication banding. Aberrant DNA replication in tumour cells and highly tumorigenic hybrids coincides with enhanced c-myc expression. In hybrids with suppressed tumorigenicity and with normal DNA replication banding c-myc expression is also reduced. Thus, a correlation between aberrant DNA replication and enhanced expression of a gene located in the same chromosomal domain is observed. Reversion of aberrant DNA replication and reduction of c-myc expression to normal in hybrid cells may be due to a site-specific trans effect which overrides the control brought about in cis by retroviral insertion near the c-myc gene.     

Distinct roles of the ERK pathway in modulating apoptosis of Ras-transformed and non-transformed cells induced by anticancer agent FR901228

Ectopic expression of oncogenic H-Ras in cells results in increases of cell susceptibility to the anticancer agent FR901228. Investigating the roles of Ras-induced pathways in FR901228-induced apoptosis, we have found that the phosphatidylinositol 3-kinase pathway plays an anti-apoptotic role, whereas the stress-activated protein kinase p38 pathway plays a pro-apoptotic role in FR901228-induced apoptosis. Interestingly, the extracellular signal-regulated kinase (ERK) pathway plays an anti-apoptotic role in non-transformed cells; however, it plays a pro-apoptotic role in Ras-transformed cells in response to FR901228 treatment. An essential role of the ERK pathway in regulating caspase-3 contents may contribute to its pro-apoptotic role in Ras-transformed cells.     

Modification of transplasma membrane oxidoreduction by SV40 transformation of 3T3 cells

Transformation of 3T3 cells by SV40 virus changes the properties of the transplasma membrane electron transport activity which can be assayed by reduction of external ferric salts. After 42 h of culture and before the growth rate is maximum, the transformed cells have a much slower rate of ferric reduction. The change in activity is expressed both by change inK _m andV _max for ferricyanide reduction. The change in activity is not based on surface charge effect or on tight coupling to proton release or on intracellular NADH concentration. With transformation by SV40 virus infection the expression of transferrin receptors increases, which correlates with greater diferric transferrin stimulation of the rate of ferric ammonium citrate reduction in transformed SV40-3T3 cells than in 3T3 cells.     

Intracellular plasminogen activator activity in growing and quiescent cells

Intracellular plasminogen activator (PA) was examined in 3T3 and transformed 3T3 cells under various growth conditions to determine whether expression of this activity changes with the growth state. During exponential growth, SV40 and benzpyrene (BP) transformed 3T3 cells exhibited 3- to 5-fold more intracellular PA activity than untransformed 3T3 cells. This relationship changed as the cells exhausted serum factors and arrested in G₁. The specific activity of intracellular PA in cells that have retained a serum-sensitive restriction point in G₁ (G₀) (3T3 and BP 3T3) increased 200- and 20-fold, respectively, at this time, while the level in cells that have lost most growth control mechanisms (SV3T3) remained constant. At confluency, 3T3 cells had considerably more PA than either of their transformed counterparts. Sparse cultures of 3T3 and BP3T3 cells arrest at G₁ following serum depravation, and also accumulate high intracellular PA activity. The addition of serum or purified epidermal growth factor to these cultures initiated cell proliferation and resulted in a rapid, actinomycin D-sensitive loss of this activity. Less than 50% of the original activity remained 30 minutes after growth stimulation. This loss of intracellular PA activity did not appear to result from the presence of serum or cellular inhibitors. Intracellular PA activity remained low following growth stimulation. It increased again as the cells traversed through G₁. These findings indicate that intracellular PA activity fluctuates with the growth state of cells, and may be related to the cell cycle. Culture conditions which place cells, whether normal or transformed, in G₁ arrest lead to increased intracellular PA, while factors that initiate growth again result in a rapid loss of this activity. This behavior is lacking in cells not subject to density-dependent inhibition of growth. Like many other correlates of transformation, comparison of intracellular PA in normal and transformed cells must be defined in terms of the growth state of the cells in question.     

Free radicals act as effectors in the growth inhibition and apoptosis of iron-treated Burkitt's lymphoma cells

The addition of ferric citrate to Burkitt's lymphoma (BL) cell lines inhibits growth, leads to the accumulation of cells in the phase G₂/M of the cell cycle and to the modulation of translocated c-myc expression. The increase in the labile iron pool (LIP) of iron-treated BL cells leads to cytotoxicity. Indeed, intracellular free iron catalyzes the formation of highly reactive compounds such as hydroxyl radicals and nitric oxide (NO) that damages macromolecular components of cells, eventually resulting in apoptosis. In this report, we have investigated the possible involvement of free radicals in the response of Ramos cells to iron. When added to Ramos cells, iron increased the intracellular levels of peroxide/peroxynitrite and NO. Moreover, the addition of free radicals scavengers (TROLOX^® and Carboxy-PTIO) neutralized the effects of iron on Ramos cells while addition of an NO donor or hydrogen peroxide (H₂O₂) to cells generated effects which partially mimicked those induced by iron addition. Collectively, our results suggest the involvement of free radicals as effectors in the iron specific growth inhibition of BL cells observed in vitro.     

Significant inhibition of hybridoma cells by exogenous application of ganglioside G M3, a possible modulator of cell growthin vitro

Gangliosides of the mouse-rat hybridoma cell line 187.1, which secretes an antibody against -light chain of mouse IgG, were isolated and structurally characterized by biochemical and immunological methods (overlay technique), and fast atom bombardment-mass spectrometry. Exclusively G _M3, substituted with C₂₄₁ and C₁₆₀ fatty acid and C₁₈₁ sphingosine, was found in this B cell derived cell line. A G _M3 (NeuGc) to G _M3(NeuAc) ratio (80 to 20), was characteristic for 187.1 cells, and absolute G _M3 amounts of about 0.3 mg 10^–9 viable cells were determined. Exogenous application of G _M3, which has been isolated from large cell preparations, to 187.1 cells showed growth inhibition in a concentration dependent manner. Using the MTT-assay and the [³H]thymidine incorporation assay, the cells exhibited a strong reduction in metabolic and proliferative activity, respectively, after exposure of cells to G _M3. G _M3 was applied in concentrations between 3M and 30M, giving evidence for strong inhibitory effects at 30M G _M3 and less but significant suppression after application of G _M3 concentrations lower than 20M. No cellular response was observed at the lowest concentration (3M) used in this study. Hybridoma cells as well as other cell types like fibroblasts, muscle cells and endothelial cells, are in general characterized by high expression of the G _M3 ganglioside, which is known to act as a modulator of cellular growth in monolayer cultures of adherent cells. Since gangliosides are released to the culture medium by cell lysis, i.e. cell death, and/or by active membrane shedding, the results obtained in this study suggest a growth regulatory role of G _M3 in high density hybridoma cell cultures.Abbreviations DMB 1,2-diamino-4,5-methylenedioxybenzene - FAB-MS fast atom bombardment-mass spectrometry - GSL(s) glycosphingolipid(s) - HPLC high performance liquid chromatography - HPTLC high performance thin layer chromatography - MTT 3,(4,5 dimethylthiazol-2-yl)2,5 diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - PBS phosphate buffered saline The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) and the nomenclature of Svennerholm (1963). Lactosylceramide or LacCer, Galß1–4Glcß1-1Cer; gangliotriaosylceramide or GgOse₃Cer; GalNAcß1–4Galß1–4Glcß1-1 Cer; gangliotetraosylceramide or GgOse₄Cer, Galß1–3GalNAcß1–4Galß1–4Glcß1-1Cer; G _M3(NeuAc), II³NeuAc-LacCer; G _M3(NeuGc), II³NeuGc-LacCer; G _M2(NeuGc), II³NeuGc-GgOse₃Cer; G _M1 or G _M1a, II³NeuAc-GgOse₄Cer; G _M1b, IV³NeuAc-GgOse₄Cer.     

Retracted: Effects of microRNA-494 on proliferation,migration, invasion,and apoptosis of medulloblastoma cells by mediating c-myc through the p38 MAPK signaling pathway

Medulloblastoma (MB) is the most prevalent brain tumor that occurs during childhood and originates from cerebellar granule cell precursors. Based on recent studies, the differential expression of several microRNAs is involved in MB, while the role of microRNA-494 (miR-494) in MB remains unclear. Therefore, we conducted this study to investigate the regulative role of miR-494 in MB cells via the p38 mitogen-activated protein kinase (MAPK) signaling pathway by mediating c-myc. In the current study, MB cells were collected and transfected with miR-494 mimic, miR-494 inhibitor, siRNA- c-myc, and miR-494 inhibitor + siRNA-c-myc. The expressions of miR-494, c-myc, p38 MAPK, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), interleukin-6 (IL-6), metadherin (MTDH), phosphatase and tensin homolog (PTEN) and survivin were determined. Cell proliferation, cell-cycle distribution, apoptosis, migration, and invasion were evaluated. The results revealed that there was a poor expression of miR-494 and high expression of c-myc in MB tissues. C-myc was determined as the target gene of miR-494. In response to miR-494 mimic, MB cells were found to have increased Bax and PTEN expressions, as well as cell number in G1 phase and cell apoptosis and decreased c-myc, p38 MAPK, Bcl-2, MTDH, IL-6, and survivin expression and cell number count in the S phase, cell proliferation, migration, and invasion. In conclusion, the results demonstrated that the upregulation of miR-494 results in the suppression of cell proliferation, migration, and invasion, while it promotes apoptosis of MB cells through the negative mediation of c-myc, which in turn inactivates the p38 MAPK pathway.     

Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells

DNA methylation is essential for mammalian development, X-chromosome inactivation, and imprinting yet aberrant methylation patterns are one of the most common features of transformed cells. One of the proposed causes for these defects in the methylation machinery is overexpression of one or more of the three known catalytically active DNA methyltransferases (DNMTs) 1, 3a and 3b, yet there are clearly examples in which overexpression is minimal or non-existent but global methylation anomalies persist. An alternative mechanism which could give rise to global methylation errors is the improper expression of one or more of the DNMTs during the cell cycle. To begin to study the latter possibility we examined the expression of the mRNAs for DNMT1, 3a and 3b during the cell cycle of normal and transformed cells. We found that DNMT1 and 3b levels were significantly downregulated in G₀/G₁ while DNMT3a mRNA levels were less sensitive to cell cycle alterations and were maintained at a slightly higher level in tumor lines compared to normal cell strains. Enzymatic activity assays revealed a similar decrease in the overall methylation capacity of the cells during G₀/G₁ arrest and again revealed that a tumor cell line maintained a higher methylation capacity during arrest than a normal cell strain. These results reveal a new level of control exerted over the cellular DNA methylation machinery, the loss of which provides an alternative mechanism for the genesis of the aberrant methylation patterns observed in tumor cells.     

Existence of a commitment program for mitosis in early G1 in tumour cells

The proliferation of normal non-tumourigenic mouse fibroblasts is stringently controlled by regulatory mechanisms located in the postmitotic stage of G₁ (which we have designated G₁ pm). Upon exposure to growth factor depletion or a lowered de novo protein synthesis, the normal cells leave the cell cycle from G₁ pm and enter G₀. The G₁ pm phase is characterized by a remarkably constant length (the duration of which is 3 h in Swiss 3T3 cells), whereas the intercellular variability of intermitotic time is mainly ascribable to late G₁ or pre S phase (G₁ ps) (Zetterberg & Larsson (1985) Proc. Natl. Acad. Sci. USA 82 , 5365). As shown in the present study two tumour-transformed derivatives of mouse fibroblasts, i.e. BPA31 and SVA31, did not respond at all, or only responded partially, respectively, to serum depletion and inhibition of protein synthesis. If the tumour cells instead were subjected to 25-hydroxycholesterol (an inhibitor of 3-hydroxy-3 methyglutaryl coenzyme A reductase activity), their growth was blocked as measured by growth curves and [³H]-thymidine uptake. Time-lapse analysis revealed that the cells were blocked specifically in early G₁ (3-4h after mitosis), and DNA cytometry confirmed that the arrested cells contained a G₁ amount of DNA. Closer kinetic analysis revealed that the duration of the postmitotic phase containing cells responsive to 25-hydroxycholesterol was constant. These data suggest that transformed 3T3 cells also contain a ‘G₁ pm program’, which has to be completed before commitment to mitosis. By repeating the experiments on a large number of tumour-transformed cells, including human carcinoma cells and glioma cells, it was demonstrated that all of them possessed a G₁ pm-like stage. Our conclusion is that G₁ pm is a general phenomenon in mammalian cells, independent of whether the cells are normal or neoplastic.     

Retinoic Acid-Induced blr1 Expression Promotes ERK2 Activation and Cell Differentiation in HL-60 Cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G₁/G₀ growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1α,25-dihydroxyvitamin D₃ and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G₁/G₀ growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.