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.     

Contact-inhibition-induced quiescent state is marked by intense nuclear expression of statin

Statin, a nuclear protein of 57,000 daltons, is found in in vitro aged, nonproliferating human fibroblasts but not in their young, replicating counterparts or transformed derivatives; it is also found in the nuclei of young fibroblasts when their growth is arrested but rapidly disappears from the cells once the restriction to growth is removed. We reported earlier that as cells leave the quiescent state, the loss of statin from the nucleus precedes the initiation of DNA synthesis; here we report that in a confluent culture, as cells leave the traverse of the replicative cycle and assume the quiescent phenotype, statin is not expressed maximally until total contact inhibition of growth is achieved. This full manifestation of statin occurs in monolayer culture with cells forming the typical swirling pattern and fibronectin organized into large intercellular cables. The late expression of statin in cells approaching the quiescent state is also verified biochemically by immunoblotting assays. The present results, taken together with those reported earlier, indicate that the nuclear appearance of statin occurs only after the complete cessation of DNA synthesis and that the full manifestation of this protein can be used as a marker for the G0 quiescent state.     

Up-regulated P21CIP1 expression is part of the regulation quantitatively controlling serum deprivation-induced apoptosis

Among the many genes which have been suggested to be required by the molecular mechanism dictating apoptotic death, some have been shown to function as pacemakers to pave the way for cells either to live or to die. Previously we have shown that immediate early gene expressions associated with the G₁ phase of cell cycle traverse are candidates for this function. Here we report that the well-known key regulator for halting cell cycling at the G₁/S border, the p21 protein known as WAF1, Cip1, Pic1, or Sdi1, is also involved in the execution of cells' suicidal death. p21 up-regulation is seen in quiescent mouse 3T3 fibroblasts stimulated to die by serum deprivation, at both message and protein levels, evidenced by increased protein presence in its targeted functional site, the nucleus. In addition, we show that this up-regulation of p21 is functionally related to the operational efficiency of the apoptotic process, in that when cells are stably transfected with an antisense construct to repress the endogenous p21-protein level, death is delayed. Quantitative protection from apoptosis with antisense p21 transfection is relatively proportional to the repressed level of this protein in the cells. Taken together, our results suggest that the apoptosis-dependent additional increase of p21 beyond the base level, seen in serum-deprived quiescent cells, may be involved in the molecular events precipitating a rapid program of cell demise, and that repression of this increase may obstruct the operation of this program and postpone the eventual death. J. Cell. Biochem. 64:434–446. © 1997 Wiley-Liss, Inc.     

Action of FR901228, a Novel Antitumor Bicyclic Depsipeptide Produced by Chromo-bacterium violaceum No. 968, on Ha-ras Transformed NIH3T3 Cells

FR901228, a novel antitumor antibiotic, reversed the transformed morphology of the Ha-ras transformants, Ras-1 cells, and inhibited their growth. The reduction of c-myc expression was observed in FR901228-treated Ras-1 cells by RNA dot-blot hybridization. This reduction of c-myc expression and morphological reversion of the transformed cells to normal were correlated with growth inhibition (G₀/G₁ arrest in cell cycle).     

Combination of Tumor Necrosis Factor Alpha and Interferon Alpha Induces Apoptotic Cell Death through a c-myc–Dependent Pathway in p53 Mutant H226br Non–Small-Cell Lung Cancer Cell Line

We investigated the role of wild-type p53 and c-myc activity in apoptosis induced by a combination of natural human tumor necrosis factor alpha (TNF-α) and natural human interferon alpha (IFN-α). Studies were performed with two human non–small-cell lung cancer cell lines, H226b, which has wild-type p53, and H226br, which has a mutant p53. The combination of IFN-α and TNF-α significantly inhibited cell growth and induced apoptotic cell death of both H226b and H226br, compared with IFN-α or TNF-α alone. Treatment with one or both cytokines did not affect the expression level of p53 in both cell lines. These results suggest that the combination of IFN-α/TNF-α induces apoptotic cell death through a p53- independent pathway. The c-myc oncogene is known to be involved in apoptosis induced by TNF. Antisense c-myc oligonucleotides have been reported to modulate cell growth or apoptosis in several cell lines. Antisense oligodeoxynucleotides were added to the culture of H226br cells before the addition of IFN-α/TNF-α. Antisense c-myc inhibited IFN-α/TNF-α cytotoxicity and apoptotic cell death. In conclusion, this study provides support for the speculation that TNF-α/IFN-α induce apoptosis through a c-myc–dependent pathway rather than a p53-dependent pathway.     

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.     

Oxidative stress-induced apoptosis in dividing fibroblasts involves activation of p38 MAP kinase and over-expression of Bax: resistance of quiescent cells to oxidative stress

Oxidative stress has been postulated to be involved in aging and age-related degenerative diseases. Cell death as a result of oxidative stress plays an important role in the age related diseases. Using human diploid fibroblasts (HDF) as model to study the mechanism of cell death induced by oxidative stress, a condition was standardized to induce apoptosis in the early passage sub-confluent HDFs by a brief exposure of cells to 250 M hydrogen peroxide. It was observed that p38 MAP kinase (MAPK) was activated soon after the treatment followed by over-expression of Bax protein in cells undergoing apoptosis. An interesting finding of the present study is that the confluent, quiescent HDFs were resistant to cell death under identical condition of oxidative stress. The contact-inhibited quiescent HDFs exhibited increased glutathione level following H₂O₂-treatment, did not activate p38 MAP kinase, or over-express Bax, and were resistant to cell death. These findings indicated that there was a correlation between the cell cycle and sensitivity to oxidative stress. This is the first report to our knowledge that describes a relationship between the quiescence state and anti-oxidative defense. Furthermore, our results also suggest that the p38MAPK activation-Bax expression pathway might be involved in apoptosis induced by oxidative stress.     

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.     

Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent

Introduction  

The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro.     

Dipeptidyl Peptidase 2 is an essential survival factor in the regulation of cell quiescence

Most cells in the body are in a resting state and undergo cell cycle progression only upon growth factor stimulation or activation. While much research on proliferation and activation has been performed, very little about signals that maintain quiescent cells in G₀ is known, preventing cell cycle entry or apoptosis. In this study, the pathways of apoptosis induction in quiescent peripheral blood cells and fibroblasts mediated by inhibition or down-regulation of Dipeptidyl Peptidase 2 (DPP2) have been explored. A decrease in DPP2 activity was found to cause resting cells to exit from G0, accompanied by a decrease in p130, p27^Kip1 and p21^Cip1 protein levels. In addition, DPP2-inhibited or down-regulated cells exhibit an increase in early G1/S progressors, with increases in the levels of retinoblastoma (pRb), p107 and cyclin D proteins. Furthermore, decrease of DPP2 activity leads to an increase in c-Myc and a decrease in Bcl-2, two events that have been associated with apoptosis induction. This apoptosis by DPP2 down-regulation is prevented in p53-/- cells or by ectopic expression of proteins that suppress p53 or c-Myc activity. Thus, DPP2 is essential for maintaining lymphocytes and fibroblasts in G₀, and its inhibition results in apoptosis mediated by induction of c-Myc and p53.     

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.     

Characterization of 57 kDa statin as a true marker for growth arrest in tissue by its disappearance from regenerating liver

Statin, a 57 kDa nuclear protein, is lost from quiescent fibroblasts in culture when they are induced to enter the cell cycle by feeding with growth factors, or by removal of contact inhibition. In order to investigate changes in statin expression during the transition from a quiescent to a cycling state in situ, we performed 70% partial hepatectomy on rats and analyzed the regenerating liver by immunofluorescence microscopy with antistatin monoclonal antibodies (S44 mAb), and by immunoblotting of liver proteins in cytoplasmic and enriched nuclear/cytoskeletal fractions. Western blot analysis showed that rat hepatocytes in situ contain a nuclear 57 kDa form of statin, as seen in cultured fibroblasts; however additional S44-immunoreactive polypeptides with molecular weights of 53 and 110 kDa are also present in both cytoplasmic and nuclear/cytoskeletal fractions. Immunofluo-rescence microscopy indicates that the proportion of S44-positive hepatocyte nuclei drops to ～60% within 24 hours after hepatectomy, a time period when re-entry of hepatocytes into the cell cycle is first observed. On Western blots of hepatocyte nuclear/cytoskeletal proteins obtained 24 hours after hepatectomy, the 57 kDa form of statin is markedly reduced. These results suggest that, although in liver the S44 antibody recognizes three proteins (53 kDa, 57 kDa, and 110 kDa), the 57 kDa in intact liver, similar to cultured fibroblasts, is the only polypeptide recognized by the statin antibody that disappears when hepatocytes are induced to re-enter the cell cycle from a quiescent state. © 1994 Wiley-Liss, Inc.     

Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity.

Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is down-regulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21(WAF/CIP)-dependent manner. To understand how mitogen-induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen-activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21(WAF/CIP) nor the tumor suppressor p53 was required for Gax-induced cell death. Arrest in G1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax-mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl-2 down-regulation and Bax up-regulation in mitogen-activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax-induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen-dependent pathways that circumvent the cell cycle to alter Bcl-2 family protein expression.     

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.     

Drosophila grim induces apoptosis in mammalian cells.

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. We show here that grim expression induces apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. We found that grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Grim-induced death is antagonized by bcl-2 in a dose-dependent manner, and neither Fas signalling nor p53 are required for grim pro-apoptotic activity. Grim protein localizes both in the cytosol and in the mitochondria of mouse fibroblasts, the latter location becoming predominant as apoptosis progresses. These results show that Drosophila grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases. These findings advance our knowledge of the mechanism by which grim induces apoptosis and show the conservation through evolution of this crucial programmed cell death pathway.     

COX-2 expression and cell cycle progression in human fibroblasts

Cyclooxygenase-2 (COX-2) is continuously expressed in mostcancerous cells where it appears to modulate cellular proliferation andapoptosis. However, little is known about the contribution oftransient COX-2 induction to cell cycle progression or programmed celldeath in primary cells. In this study we determined whether COX-2regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E₂(PGE₂) were not detected in quiescent cells but wereexpressed during the G₀/G₁ phase of the cellcycle induced by serum. Inhibition of COX-2 did not alter G₀/G₁ to S phase transition or induceapoptosis at concentrations that diminished PGE₂.Addition of interleukin-1 to serum enhanced COX-2 expression andPGE₂ synthesis over that by serum alone but had no effecton the progression of these cells into S phase. Furthermore,platelet-derived growth factor drove the G₀ fibroblasts into the cell cycle without inducing detectable levels of COX-2 orPGE₂. Collectively, these data show that transient COX-2expression in primary human fibroblasts does not influence cell cycle progression.

     

Rapid induction of competence formation is PDGF-isoform specific.

Platelet-derived growth factor (PDGF) stimulates the expression of a number of genes associated with entry of quiescent Balb/c-3T3 fibroblasts into the cell cycle. We determined that two of these genes, c-myc and c-fos, are induced equivalently in medium supplemented with platelet-poor plasma (PPP) and either PDGF-BB or PDGF-AA. The rate at which fibroblasts entered S phase was also similar in PDGF-BB- and AA-treated cells as was the expression of the late G1 gene, thymidine kinase (TK). However, PDGF-AA must be present for a period of 16 h to stimulate the proliferation of 90% of the cells, whereas PDGF-BB was required for only 4 h. Exposure of cells to PDGF-AA for 4 h, a time during which maximum expression of c-fos and c-myc occurred, only induced 20% of the cells in a quiescent population to enter the cell cycle. Therefore, PDGF-AA-mediated expression of the immediate early genes c-fos and c-myc may be necessary but is not sufficient to rapidly stimulate density-arrested Balb/c-3T3 fibroblasts into the competent state. Thus, these data suggest that PDGF-AA and PDGF-BB initiate traverse of the cell cycle by distinct mechanisms.