Apoptosis In Primary Cultures Research Articles

Choline deficiency induces apoptosis in primary cultures of fetal neurons

Choline deficiency induces apoptosis in primary cultures of fetal neurons

Selenium and signal transduction: roads to cell death and anti-tumour activity.

Accumulated evidence from prospective studies, intervention trials and studies on animal models of cancer have suggested a strong inverse correlation between selenium intake and cancer incidence. Several putative mechanisms have been suggested to mediate the chemopreventive activities of selenium: of these, the inhibition of cellular proliferation and the induction of apoptosis are particularly attractive. The mitogen activated protein kinase (MAPK) pathways are known to be important regulators of cell death and our recent work has focused on the involvement of these pathways in selenium-induced apoptosis in primary cultures of oral cancers and corresponding normal mucosa derived from biopsy material. Using this system, the oral carcinoma cells were found to have enhanced sensitivity to apoptosis when treated with certain selenium compounds compared to normal oral mucosa. Induction of Fas ligand was associated with selenium-induced apoptosis. Signal transduction studies suggests that selenium induces several changes in the MAPK signalling pathways but functional intervention/inhibitor studies indicate that activation of the JNK pathway seems to be most important.

Roles of BCL-2 and caspase 3 in the adenosine A3 receptor-induced apoptosis.

Selective A3 adenosine receptor agonists have been shown to induce apoptosis in a variety of cell types. In this study we examined the effects of adenosine receptor agonists selective for A1, A2A, or A3 receptors on the induction of apoptosis in primary cultures of rat astrocytes and in C6 glial cells. Treatment of the cells with the A3 receptor agonist Cl-IB-MECA (10 microM) induced apoptosis in both cell types. The effects of Cl-IB-MECA were partially antagonized by the A3 receptor-selective antagonist MRS 1191. In contrast, the A1 and A2A receptor agonists, CPA and CGS 21680, respectively, did not have significant effects on apoptosis in these cells. Cl-IB-MECA reduced the expression of endogenous Bcl-2, whereas it did not affect the expression of Bax. Overexpression of Bcl-2 in C6 cells abrogated the induction of apoptosis induced by the A3 agonist. Cl-IB-MECA also induced an increase in caspase 3 activity and caspase inhibitors decreased the apoptosis induced by the A3 agonist. These findings suggest that intense activation of the A3 receptor is pro-apoptotic in glial cells via bcl2 and caspase-3 dependent pathways.

Hypoxic conditions restore lost sensitivity to the growth inhibitory effect of Transforming Growth Factor beta-1 (TGF beta-1) in proliferating rat hepatocytes in vitro

TGF beta-1 is known to be a growth inhibitor of regenerating liver, and an inducer of hepatocyte apoptosis in primary culture. However, hepatocytes can proliferate after partial hepatectomy even at high serum TGF beta-1 concentrations. In this study we used the primary cultures of rat hepatocytes for 10 days to investigate how TGF beta-1 affects proliferating hepatocytes. DNA synthesis peaked on day 8 of culture, and TGF beta-1-induced apoptosis was significantly suppressed on day 8 compared to days 2, 5, and 10. Flow-cytometric analysis revealed that hepatocytes that had incorporated BrdU were resistant to the apoptotic effect of TGF beta-1, and Northern blot analysis showed that TGF beta receptor mRNA was down-regulated on day 8. Hypoxic conditions restores TGF beta receptor mRNA expression and the lost sensitivity of proliferating hepatocyte to TGF beta-1.

Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells.

Apoptosis of arterial cells induced by oxidized low density lipoproteins (OxLDL) is thought to contribute to the progression of atherosclerosis. However, most data on apoptotic effects and mechanisms of OxLDL were obtained with extensively oxidized LDL unlikely to occur in early stages of atherosclerotic lesions. We now demonstrate that mildly oxidized LDL generated by incubation with oxygen radical-producing xanthine/xanthine oxidase (X/XO) induces apoptosis in primary cultures of human coronary endothelial and SMC, as determined by TUNEL technique, DNA laddering, and FACS analysis. Apoptosis was markedly reduced when X/XO-LDL was generated in the presence of different oxygen radical scavengers. Apoptotic signals were mediated by intramembrane domains of both Fas and tumor necrosis factor (TNF) receptors I and II. Blocking of Fas ligand (FasL) reduced apoptosis by 50% and simultaneous blocking of FasL and TNF receptors by 70%. Activation of apoptotic receptors was accompanied by an increase of proapoptotic and a decrease in antiapoptotic proteins of the Bcl-2 family and resulted in marked activation of class I and II caspases. Mildly oxidized LDL also activated MAP and Jun kinases and increased p53 and other transcription factors (ATF-2, ELK-1, CREB, AP-1). Inhibitors of Map and Jun kinase significantly reduced apoptosis. Our results provide the first evidence that OxLDL-induced apoptosis involves TNF receptors and Jun activation. More important, they demonstrate that even mildly oxidized LDL formed in atherosclerotic lesions may activate a broad cascade of oxygen radical-sensitive signaling pathways affecting apoptosis and other processes influencing the evolution of plaques. Thus, we suggest that extensive oxidative modifications of LDL are not necessary to influence signal transduction and transcription in vivo.

Brefeldin A induces p53-independent apoptosis in primary cultures of human prostatic cancer cells.

The objective of this study was to investigate growth-inhibitory and apoptotic activity of the experimental antitumor drug, brefeldin A (BFA), on primary cultures of human epithelial cells derived from prostatic adenocarcinomas. Clonal assays were performed to evaluate the effects of BFA on growth of prostatic cancer cell strains. Loss of cell viability in response to BFA was assessed by trypan blue exclusion. Induction of apoptosis by BFA was evaluated by morphologic criteria, electrophoretic assay of DNA fragmentation, and a cell death ELISA. Immunoblots were used to monitor p53 and pRB expression in response to BFA. BFA was growth-inhibitory at a half-maximal concentration of 5 ng./ml. (18 nM). Morphological manifestations of apoptosis were evident by 24 hours of treatment. Cell viability declined and the cell death ELISA indicated an 18-fold increase in apoptosis in BFA-treated versus untreated cells at 48 hours. DNA fragmentation was also seen at 48 hours. Levels of p53 were not altered by BFA, but pRB was maintained in a hypophosphorylated state by BFA treatment. BFA is a potent inducer of apoptosis in prostatic cancer cells via a p53-independent mechanism. Cells derived from low-grade as well as high-grade cancers responded similarly to BFA. Since p53-mediated pathways of apoptosis may frequently be abrogated in prostatic cancer cells, agents such as BFA that induce p53-independent cell death may be promising candidates for chemotherapeutic agents.

Staurosporine-induced apoptosis in cultured chick embryonic neurons is reduced by polyethylenimine of low molecular weight used as a coating substrate.

The survival of neurons largely depends on adhesion to extracellular matrix proteins. This study investigated the influence of polycationic macromolecules of different molecular weights used as coating substrates on apoptosis in primary cultures of chick embryonic neurons. Coating of the culture flasks with positively charged polyethylenimine (PEI) of 12, 32 and 1616 kDa led to different susceptibilities of the neurons to apoptosis induced by staurosporine and serum deprivation. In cultures grown as usual on polylysine (PL)-coated flasks, we found, after 24 h of incubation in medium with serum (controls), serum-free medium and staurosporine (200 nM)-containing serum-free medium, 15, 35 and 63% apoptotic neurons, respectively as evaluated by nuclear staining with Hoechst 33258. Using 12-kDa PEI as a coating substrate, only 11, 15 and 47% apoptotic neurons could be determined in controls, serum-deprived and staurosporine-treated cultures, respectively. No change in the percentage of apoptotic neurons was found after 24 h of serum deprivation or treatment with staurosporine in cultures grown on 32-kDa PEI compared with cultures grown on PL. However, in staurosporine-treated cultures grown on 1616-kDa PEI, the percentage of apoptotic neurons was even higher than in cultures grown on PL. Immunostaining using neurofilament (NF) antibodies revealed that the reduction of staurosporine-induced apoptosis using 12-kDa PEI instead of PL as a coating substrate was accompanied by a reduced disruption and aggregation of the neurofilaments. Thus, the usage of the newly synthesized 12-kDa PEI as a coating substrate enhanced neuronal resistance to apoptosis.

Apoptosis in Primary Cultures of E14 Rat Ventral Mesencephala: Time Course of Dopaminergic Cell Death and Implications for Neural Transplantation

Transplantation using fetal nigral grafts has been performed by various groups worldwide in over 200 Parkinson's disease (PD) patients in an attempt to restore dopaminergic (DA) input to the striatum. However, the proportion of the implanted DA neurons that survives, whether using suspension, partially dissociated, or solid grafts, is small, often as low as 5 to 10%, which is insufficient to allow a full functional recovery. A significant proportion of the transplanted neurons in animal models of PD has been shown to die via apoptosis, but the reason for this is unclear. Since the methods used to prepare donor tissue for neural transplantation and in vitro culture are identical, we have looked at the time course of DA neuron loss following cell suspension preparation using an in vitro assay system and considered whether the procedures used may, in part, be responsible for the poor DA neuron survival. Primary dissociated cultures of E14 rat ventral mesencephala were incubated for different periods in serum-containing and serum-free media. After fixation, the TUNEL method, as well as ethidium bromide and acridine orange, were used to detect apoptosis, and DA neurons were localized immunocytochemically. Results showed that most apoptosis occurred during the first 24 h and that 50% of the DA neurons were lost in the first 8 h. Double-immunofluorescent labeling confirmed the presence of TUNEL+ve nuclei within DA neurons. There was no difference in either the extent or rate of loss between the serum-containing and serum-free medium during the first 32 h. We suggest, therefore, that existing methods used to prepare cell suspensions probably induce apoptosis and may need to be modified in order to increase the survival of DA neurons.

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture.

The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 microM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 microM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.

Metabotropic glutamate receptor agonists protect from oxygen–glucose deprivation- and colchicine-induced apoptosis in primary cultures of cerebellar granule cells

The effects of the metabotropic glutamate receptor agonists against apoptosis induced by oxygen–glucose deprivation or colchicine were studied in the primary cultures of mature cerebellar granule cells. Oxygen–glucose deprivation (90 min) or addition of colchicine (1 μM) resulted in neuronal damage as revealed by Trypan Blue assay 12 h later. Further analysis demonstrated that the cells exposed to oxygen–glucose deprivation or colchicine exhibit typical features of apoptosis: internucleosomal DNA fragmentation, condensation and fragmentation of chromatin and typical DNA ladder on agarose gel electrophoresis. Metabotropic glutamate receptor agonist, (1S,3R)-1-aminocycloheptane- trans-1,3-dicarboxylic acid, acting at group I and II receptors, and selective agonist, (2S,1′R,2R′,3R′)-2(2,3-dicarboxycyclopropyl)glycine, acting at group II receptors, added to cells recovering from oxygen-glucose deprivation exerted neuroprotective action against oxygen–glucose deprivation-induced apoptosis. Similar neuroprotective effects of metabotropic glutamate receptor agonists were observed against colchicine-induced apoptosis. The results thereby provide evidence that metabotropic glutamate receptor agonists have therapeutic potential in the treatment of pathologies associated with increased neuronal apoptosis. The selective protein kinase C inhibitor bisindolylmaleimide (100 nM) abolished the neuroprotective action of (1S,3R)-1-aminocycloheptane- trans-1,3-dicarboxylic acid, whereas the activator of adenylyl cyclase forskolin (10 μM) abolished the neuroprotective action of (2S,1′R,2R′,3R′)-2(2,3-dicarboxycyclopropyl)glycine (30 μM) against colchicine-induced apoptosis. It is concluded that both phosphoinositide hydrolysis with consequent activation of protein kinase C and inhibition of adenylyl cyclase seem to contribute to the neuroprotective action of metabotropic glutamate receptor agonists against neuronal apoptosis in the primary culture of cerebellar granule cells.

Changes in Tissue Transglutaminase Activity and Expression during Retinoic Acid-Induced Growth Arrest and Apoptosis in Primary Cultures of Human Epithelial Prostate Cells

Changes in Tissue Transglutaminase Activity and Expression during Retinoic Acid-Induced Growth Arrest and Apoptosis in Primary Cultures of Human Epithelial Prostate Cells

Cyclic stretch induces both apoptosis and secretion in rat alveolar type II cells

We examined the effects of short-term cyclic stretch on both phosphatidylcholine (PC) secretion and apoptosis in primary cultures of rat alveolar type II cells. A 22% cyclic stretch (3 cycles/min) was applied to type II cells cultured on silastic membranes using a Flexercell strain unit. This induced, after a lag period of about 1 h, a small, but significant release of [ 3H]PC from prelabelled cells. In addition, stretch increased nuclear condensation, the generation of oligosomal DNA fragments and the activation of caspases. Similar responses were triggered by sorbitol-induced osmotic shock, but not by the secretagogue ATP. We conclude that stretch can induce both apoptosis and PC secretion in alveolar type II cells and propose that these diverse responses occur within the lung as a consequence of normal respiratory distortion of the alveolar epithelium.

Changes in tissue transglutaminase activity and expression during retinoic acid-induced growth arrest and apoptosis in primary cultures of human epithelial prostate cells.

We treated primary epithelial cells from human normal prostate (NEPC) and prostate cancer (CEPC) with all-trans-retinoic acid (RA) to study whether it regulates the activity of tissue transglutaminase (tTGase), an enzyme that accumulates in cells undergoing apoptosis. tTGase activity was assessed by [14C]spermidine incorporation; tTGase, P53, Bcl-2, and p21 protein levels were evaluated by Western blotting; and RA receptors (RAR alpha, -beta, and -gamma), tTGase, retinol-binding protein (RBP), and cellular RBP type I transcripts were determined by semiquantitative RT-PCR. After 72-96 h of 10(-6) mol/L RA treatment, cell growth inhibition and apoptosis were associated with increased tTGase activity in both NEPC and CEPC, and with increased tTGase protein and messenger ribonucleic acid levels only in NEPC. Moreover, RA down-regulated RAR alpha and -beta and increased RBP messenger ribonucleic acid levels in NEPC, whereas it increased RAR beta gene expression and decreased Bcl-2 protein levels in CEPC. Our results suggest that RA induces tTGase gene expression and enzyme activity in normal prostate cells, and that RA-regulated pathways are impaired in cancer cells. Moreover, down-regulation of Bcl-2 protein and up-regulation of RAR beta suggest that retinoid may act on the genetic defect responsible for prostate cancer progression.

Anthraquinone cytotoxicity and apoptosis in primary cultures of rat hepatocytes

We compared three different anthraquinones, rhein (4,5-dihydroxy-anthraquinone-2-carboxylic acid), danthron (1,8-dihydroxy-anthraquinone) and chrysophanol (1,8-dihydroxy-3-methylanthraquinone), with respect to their toxicity and ability to induce apoptosis in primary cultures of rat hepatocytes. Rhein was the most effective in producing free radicals, and was the only one of the tested anthraquinones that could induce apoptosis. Addition of 50μM rhein to hepatocyte cultures led to depletion of intracellular reduced glutathione (GSH) and ATP and accumulation of lipid peroxidation products. The substances N,N′-diphenyl-p-phenylenediamine (DPPD), dithiothreitol (DTT), nifedipine and desferal all protected the hepatocytes, i.e. prevented viability loss and ATP depletion, and decreased the GSH depletion.Cultures exposed to rhein for 15min and subsequently rinsed and incubated for 16h under normal culture conditions (complete medium) exhibited apoptosis, as shown by DNA fragmentation, nuclear condensation and positive TUNEL reaction. Pretreatment with the antioxidant DPPD and the iron-chelator desferal gave complete protection against apoptosis.No signs of oxidative cell damage were detected when the cultures were exposed to danthron or chrysophanol. All three anthraquinones did, however, cause an immediate increase in the intracellular Ca2+ concentration.We conclude that rhein, which contains one carboxyl group, is a suitable substrate for one-electron-reducing enzymes and an effective redox cycler, which leads to the production of oxygen-derived free radicals that eventually induce apoptotic cell death.

The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis.

Phenobarbital and clofibrate, two non-genotoxic carcinogens, have been investigated regarding the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis in primary cultures of rat hepatocytes. Low toxicity concentrations, 200 and 100 microg/ml for phenobarbital and clofibrate respectively, were used to examine their effect on spontaneous or transforming growth factor beta1 (TGFbeta1)-induced apoptosis and on the expression of antioxidant defence enzymes (superoxide dismutases and catalase). The increased incidence of apoptotic nuclei was visualized in TGFbeta1-treated cultures with the fluorescent dye Hoechst 33258 and was quantified under all experimental conditions by measurement of the hypodiploid peak in DNA histograms obtained by flow cytometry. Both substances, when added separately to hepatocyte cultures and incubated for 24 and 48 h, significantly diminished spontaneous apoptosis and exhibited a slight suppression of TGFbeta1-induced apoptosis. Endogenous peroxide production by hepatocytes increased with TGFbeta1, phenobarbital or clofibrate and the increase was greater with phenobarbital and in the presence of TGFbeta1 with both drugs. Gene expression of catalase and Mn- and Cu,Zn superoxide dismutases (SOD) was evaluated by northern blot analysis of hepatocytes incubated in the presence of phenobarbital or clofibrate with or without TGFbeta1 and the following differences were detected: phenobarbital induced a significant decrease in both dismutases (to 56%, P < 0.05, and 55%, P < 0.05, for Mn- and Cu,Zn-SOD respectively) and a 2-fold increase (P < 0.01) in catalase; clofibrate induced a slight decrease in both SODs and a 4-fold increase (P < 0.05) in catalase; TGFbeta1 significantly decreased to 37% (P < 0.05) expression of catalase while not significantly affecting expression of both SODs. We conclude that inhibition of spontaneous apoptosis induced by either phenobarbital or clofibrate is accompanied by increases in the endogenous levels of peroxides and by significant induction of catalase gene expression. Furthermore, the lack of effect of both compounds on TGFbeta1-induced apoptosis could be a consequence of the inability of these two compounds to counteract the depressing effect of TGFbeta1 on expression of catalase.

The coordinate regulation of DNA synthesis and suppression of apoptosis is differentially regulated by the liver growth agents, phenobarbital and methylclofenapate.

The coordinate regulation of DNA synthesis and suppression of apoptosis was investigated in a rat hepatocyte cell culture system which supports high level induction of DNA synthesis by the peroxisome proliferator, methylclofenapate (MCP) (Plant, N.J. et al., 1998, Carcinogenesis, 19, 925-931). The peroxisome proliferators are hepatocyte mitogens in chemically defined media: glucocorticoid-induced PPARalpha is linked to peroxisome proliferator mitogenesis (Plant, N.J. et al., 1998, Carcinogenesis, 19, 925-932). Phenobarbital (PB) induced moderate induction of DNA synthesis (200-300% of control), but the peak of induction was 40 h after treatment. In hepatocytes that had undergone DNA synthesis, PB increased the proportion of binucleates by 200-300%. Both PB and MCP were able to suppress apoptosis in a dose-dependent manner, while the endogenous mitogen epidermal growth factor failed to suppress apoptosis. The suppression of apoptosis by MCP was reversible; withdrawal of MCP led to rapid induction of apoptosis. The presence of hydrocortisone is required for suppression of apoptosis by peroxisome proliferators, but not for PB. MCP failed to suppress apoptosis in primary cultures of guinea-pig hepatocytes. Comparison of the stability of hepatocytes labelled with bromodeoxyuridine (BrdUrd) and [3H]thymidine revealed that approximately 40% of cells labelled with BrdUrd were lost over a period of 14 days, whereas cells labelled with thymidine remained stable over this period. Hepatocytes were therefore treated with MCP, labelled with [3H]thymidine, maintained for 14 days, and peroxisome proliferator withdrawn. While the apoptotic index in unlabelled cells was 1.7%, no apoptosis was detected in labelled cells. In order to compare the mechanism of suppression of apoptosis, hepatocytes were cultured in the presence of either PB or MCP for 14 days. When MCP was substituted for PB in cells cultured in the presence of PB, the monolayer was maintained, but when PB was used to replace MCP in cells cultured in the presence of MCP, the monolayer of hepatocytes degenerated rapidly. The results demonstrate mechanistic differences in the coordinate regulation of cell growth and apoptosis in hepatocytes by PB and MCP.

In vitro and in vivo effects of an extract of Ginkgo biloba (EGb 761), ginkgolide B, and bilobalide on apoptosis in primary cultures of rat hippocampal neurons

Primary cultures of rat hippocampal neurons were prepared and exposed to increasing concentrations of a peroxyl radical-generator, 2,2′-azobis 2 amidinopropane (AAPH). Addition of AAPH (20 or 50 mM) to the medium caused a decrease in cell viability and an increase in the number of apoptotic cells. Values for the number of nucleosomes were obtained using an ELISA technique. “Factor F,” an indicator of enrichment in nucleosomes, was found to be directly proportional to the number of neuronal apoptoses. Addition of an extract of Ginkgo biloba (Egb 761; 5–20 μg/ml) or ginkgolide B (one of its terpenoid constituents; 0.2 or 0.4 μg/ml) to the culture medium in vitro led to increases in cell viability and decreases in the number of hippocampal cells undergoing AAPH-induced apoptosis, whereas addition of bilobalide (another terpenoid constituent of Egb 761; 0.1–1.0 μg/ml) was ineffective. These in vitro results were corroborated and extended when these same substances were administered to rats in vivo. Oral administration of Egb 761 (50 mg/kg/day) for 8 days caused a significant increase in cell viability and a highly significant decrease in the numbers of both spontaneously occurring and AAPH-induced apoptoses. Similar protective effects were observed with ginkgolide B (2 mg/kg/day, p.o.), whereas bilobalide (2 mg/kg/day, p.o.) was ineffective. As AAPH enhances the production of peroxyl radicals, the protective actions of subacute in vivo treatments with Egb 761 and ginkgolide B appear to be associated with an anti-lipoperoxidative effect of these substances. Drug Dev. Res. 45:23–29, 1998. © 1998 Wiley-Liss, Inc.

P53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (-/-) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53-/- mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53-/- cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53-/- cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.

Differential inducibilities of GFAP expression, cytostasis and apoptosis in primary cultures of human astrocytic tumours.

Glial fibrillary acidic protein (GFAP) is an astrocytic lineage-specific intermediate filament protein, and its expression or non-expression is inversely correlated with the tumourigenecity of astrocytoma cells. To estimate the GFAP levels of astrocytes in intracranial tumour tissues, we established primary cultures from six astrocytic tumour specimens and used a double-staining flow cytometric method to detect the different levels of GFAP among these primary cultures. Although these primary cultures exhibited the same Matrigel invasiveness, their GFAP expression is inversely related to the rate of cell growth and the histologic grade of the original tumour. Phenylacetate, 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, which are potent inducers of differentiation in various cancer cells, have been examined for their effects on these primary cultures. Cytostasis was more or less caused by these compounds in all six primary cultures, but induction of GFAP was observed only in the primary culture derived from a less malignant astrocytoma specimen having the highest intrinsic GFAP level. Interestingly, this primary culture, but not others, also exhibited increased HRG-alpha expression after phenylacetate or sodium butyrate treatment. Loss of the inducibility of differentiation-related gene expression could be one of the events involved in the malignant progression of astrocytomas. In addition, the chemotherapeutic agent BiCNU has a killing effect on all six primary culture cells, with LD50 less than 60 nM. The underlying mechanism was through the induction of apoptosis in these primary culture cells regardless of their varying malignancies of original tumours. However, unlike colon cancer and leukaemia cells, sodium butyrate could not induce apoptosis within 4 days in these astrocytic tumour cells, indicating that the cell context of different cell types indeed determined the ability of sodium butyrate to induce apoptosis.

Effect of phorbol ester (PMA) on antioxidant enzyme expression in TGF-beta 1-induced apoptosis in primary cultures of hepatocytes.

Apoptosis has been documented as a fundamental component of the life cycle of many cell types. One of the characteristics of this process is the cleavage of genomic DNA into oligonucleosomal fragments. The multifunctional cytokine TGF-beta 1 has been described to induce apoptosis in cultured hepatocytes although in this condition DNA fragmentation has not been detected. We investigated whether TGF-beta 1-induced apoptosis was associated with DNA fragmentation and was affected by PMA. Agarose gel electrophoresis of TGF-beta 1-treated hepatocytes shows a typical ladder-like pattern of DNA fragments and PMA, a selective stimulator of protein kinase C, diminishes the DNA fragmentation and cell death. It has been described that the antioxidant enzyme systems play an important role in the control of apoptosis and that the apoptogenic ability of TGF-beta 1 is through the inhibition of antioxidant enzyme expression in cultured hepatocytes [8]. However, PMA does not induce significant changes levels of manganese superoxide dismutase, copper-zinc superoxide dismutase and catalase mRNAs. Our data reveal that the attenuation of TGF-beta 1-induced DNA fragmentation by PMA is not associated with changes in the expression of antioxidant systems and is probably due selectively to the stimulation of protein kinases C.