5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.     

MAPK (ERK2) kinase--a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth.

Limited clinical and experimental studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) may inhibit gastric cancer growth. However, the mechanisms involved are not completely understood and cannot be explained by COX-2 inhibition alone. MAPK signaling pathway is essential for cell proliferation, but the effect of NSAIDs on MAPK activity and phosphorylation in gastric cancer has never been studied. Since increased and unregulated cell proliferation and reduced cell apoptosis are important features of cancer growth, we studied whether NS-398, a selective COX-2 inhibitor and/ or indomethacin (IND), a non-selective NSAID: 1) inhibit gastric cancer cell proliferation, 2) whether this inhibition is mediated via MAPK (ERK2), and 3) whether NSAIDs enhance apoptosis in gastric cancer cells. Human gastric epithelial cells (MKN28) derived from gastric tubular adenocarcinoma were cultured and treated with either vehicle, IND (0.25-0.5mM) or NS-398 (50-100 microM) for 6, 16, 24 and 48h. Studies: 1) Cellular proliferation was determined by 3H-thymidine uptake. 2) MAPK activity was measured by incorporation of radiolabeled phosphate into myelin basic protein. 3) Apoptosis was evaluated using TUNEL assay. IND and NS-398 significantly inhibited the proliferation of MKN28 cells at 24h by 3.5 - 5 fold (p<0.002) and at 48h by 2.5 - 10 fold (p<0.02). Both NSAIDs also significantly inhibited ERK2 activity: IND >53% inhibition, NS-398, 100 microM >72% inhibition; all p<0.05. Both IND and NS-398 significantly increased apoptotic index. In conclusion, IND and NS-398 significantly inhibit proliferation and growth of human gastric cancer cell line MKN28. This effect is mediated by NSAID-induced inhibition of MAPK (ERK2) kinase signaling pathway, essential for cell proliferation. NSAIDs also increase apoptosis in MKN28 cells. In addition to inhibiting cyclooxygenase, NSAIDs inhibit phosphorylating enzymes--kinases essential for signaling cell proliferation.     

Screening for antiproliferative effects of cellular components from lactic acid bacteria against human cancer cell lines

Whole cells, cytoplasms and peptidoglycans of ten different lactic acid bacteria (LAB) were tested for in vitro cytotoxicity on diverse cancer cell lines using the ³H-thymidine incorporation assay. The peptidoglycans and cytoplasm fractions, as well as heat-killed whole cells of LAB, had significant antiproliferative activities against several cancer cell lines. In particular, the cytoplasm fractions exhibited marked direct antiproliferative activities against colon and gastric cancer cell lines, whereas the peptidoglycans retarded growth of colon and bladder cancer cell lines. The cytoplasm fractions of Bifidobacterium longum and Lactococcus lactis ssp. lactis inhibited proliferation of two cancer cell lines by 50% at 33 and 23 g ml^–1 for SNUC2A (a human colon adenocarcinoma cell line) and 17 and 11 g ml^–1 for SNU-1 (a human gastric cancer cell line), respectively.     

Pregnenolone Protects Mouse Hippocampal (HT-22) Cells against Glutamate and Amyloid Beta Protein Toxicity

In the present work we have examined whether the neurosteroid pregnenolone has any neuroprotective effects against glutamate and amyloid beta protein neurotoxicity using immortalized clonal mouse hippocampal cell line (HT-22). The neurosteroid pregnenolone protects HT-22 cells against both 5 mM glutamate and 2 M amyloid beta protein induced cell death in a concentration dependent manner. Optimum protection was attained at 500 nM pregnenolone, against both 5 mM glutamate as well as 2 M amyloid beta protein induced HT-22 cell death. Furthermore, using confocal immunoflourescence microscopy we observed that 20 hours of treatment with 5 mM glutamate resulted in intense nuclear localization of the glucocorticoid receptor (GR) in HT-22 cells as compared to control untreated cells. Interestingly, 500 nM pregnenolone treatment for 24 hours, followed by 20 hours treatment with 5 mM glutamate resulted in dramatic reduction in GR nuclear localization. These results show that (i) pregnenolone has neuroprotective effects against both glutamate and amyloid beta protein neuropathology and (ii) prevention of glucocorticoid receptor (GR) localization to the nucleus may be involved in the observed neuroprotective effects of pregnenolone against glutamate neurotoxicity.     

Antithyroid cancer effects of human neural stem cells expressing therapeutic genes on anaplastic thyroid cancer cells

Stem cells that express therapeutic proteins have been identified to have an anticancer effects on various types of cancer. In the present study study, human neural stem cells (hNSCs) that were genetically engineered to express cytosine deaminase (CD) and human interferon-β (IFN-β) were used for anaplastic thyroid cancer (ATC) treatment owing to their tumor-tropic properties and therapeutic effects. CD is an enzyme that converts 5-fluorocytosine (5-FC), a prodrug, to 5-fluorouracil (5-FU) which is a medication to suppress tumor growth through DNA synthesis inhibition. Also, IFN-β suppresses tumor growth by the induction of apoptotic process. In water soluble tetrazolium salt (WST) assay, SNU-80 cells which are human female ATC cells were cocultured with three cell types including engineered hNSCs such as HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-β cells on transwells and treated with 5-FC for 72 hours. Finally, the SNU-80 cell viability was reduced by the coculture with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In dichlorofluorescein diacetate (DCF-DA) and TdT-mediated dUTP nick-end labeling (TUNEL) assays, the production of reactive oxygen species (ROS) and the number of apoptotic cells were increased by HB1.F3.CD and HB1.F3.CD.IFN-β cells in the presence of 5-FC. In Western blot assay, ROS, and apoptosis-related genes were increased in SNU-80 cells when they were cocultured with HB1.F3.CD and HB1.F3.CD.IFN-β cells. In transwell migration assay, hNSCs selectively migrated to SNU-80 cells because hNSCs interacted with chemoattractant factors like SDF-1α, uPAR, and CCR2 secreted by SNU-80 cells. Taken together, engineered hNSCs were revealed to selectively migrate to ATC cells and to inhibit growth as well as to induce apoptosis of ATC cells via ROS production through the actions of transgenes such as CD and IFN-β. Therefore, these engineered hNSCs can be promising candidates for the treatment of metastatic ATC.     

Bcl-2, survivin and variant CD44 v7-v10 are downregulated and p53 is upregulated in breast cancer cells by progesterone: Inhibition of cell growth and induction of apoptosis

Progesterone inhibits the proliferation of normal breast epithelial cells in vivo, as well as breast cancer cells in vitro. But the biologic mechanism of this inhibition remains to be determined. We explored the possibility that an antiproliferative activity of progesterone in breast cancer cell lines is due to its ability to induce apoptosis. Since p53, bcl-2 and survivin genetically control the apoptotic process, we investigated whether or not these genes could be involved in the progesterone-induced apoptosis.We found a maximal 90% inhibition of cell proliferation with T47-D breast cancer cells after exposure to 10 M progesterone for 72 h. Control progesterone receptor negative MDA-231 cancer cells were unresponsive to 10 M progesterone. The earliest sign of apoptosis is translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane and can be monitored by the calcium-dependent binding of annexin V in conjunction with flow cytometry. After 24 h of exposure to 10 M progesterone, cytofluorometric analysis of T47-D breast cancer cells indicated 43% were annexin V-positive and had undergone apoptosis and no cells showed signs of cellular necrosis (propidium iodide negative). After 72 h of exposure to 10 M progesterone, 48% of the cells had undergone apoptosis and 40% were annexin V positive/propidium iodide positive indicating signs of necrosis. Control untreated cancer cells did not undergo apoptosis. Evidence proving apoptosis was also demonstrated by fragmentation of nuclear DNA into multiples of oligonucleosomal fragments.After 24 h of exposure of T47-D cells to either 1 or 10 M progesterone, we observed a marked down-regulation of protooncogene bcl-2 protein and mRNA levels. mRNA levels of survivin and the metastatic variant CD44 v7-v10 were also downregulated. Progesterone increased p53 mRNA levels.These results demonstrate that progesterone at relative high physiological concentrations, but comparable to those seen in plasma during the third trimester of human pregnancy, exhibited a strong antiproliferative effect on breast cancer cells and induced apoptosis.     

Cadmium-induced apoptosis in C6 glioma cells: Mediation by caspase 9-activation

The induction of apoptotic cell death by cadmium was investigated in eight mammalian cell lines. Great differences in the cytotoxicity of cadmium were found with different cell lines: Rat C6 glioma cells turned out to be most sensitive with an IC₅₀-value of 0.7 M, while human A549 adenocarcinoma cells were relatively resistant with an IC₅₀-value of 164 M CdCl₂. The mode of cadmium-induced cellular death was identified to involve apoptotic DNA fragmentation in three cell lines, i.e., in C6 glioma cells, E367 neuroblastoma cells and NIH3T3 fibroblasts. In C6 glioma cells, this process was investigated in detail. Internucleosomal DNA-fragmentation occurred 40 h after application of CdCl₂ and was concentration-dependent between 1–100 M CdCl₂, followed by a decrease at higher concentrations due to necrotic processes. Apoptotic chromatin-condensation and nuclear fragmentation was observed 48 h after application of 2.5 M CdCl₂. Furthermore, cadmium (1 M, 48 h) caused a breakdown of the mitochondrial membrane potential as shown by the decline in mitochondrial uptake of rhodamine 123. Also, we found an activation of caspase 9, a protease known to be activated in apoptotic processes following mitochondrial damage. Besides Cd²⁺, other toxic heavy metal ions (Hg²⁺, Pb²⁺, Ni²⁺, Fe²⁺, CrO₄ ^2–, Cu²⁺ or Co²⁺) did not induce apoptotic DNA fragmentation in C6 cells. The only exception was Zn²⁺ which caused apotosis at high concentrations (>150 M) whereas it protected against cadmium-induced apoptosis at low concentrations (10–50 M).     

Inhibition of gastric cancer cell proliferation by resveratrol: role of nitric oxide

Resveratrol is a dietary phytochemical that has been shown to inhibit proliferation of a number of cell lines, and it behaves as a chemopreventive agent in assays that measure the three stages of carcinogenesis. We tested for its chemopreventive potential against gastric cancer by determining its interaction with signaling mechanisms that contribute to the proliferation of transformed cells. Low levels of exogenous reactive oxygen (H(2)O(2)) stimulated [(3)H]thymidine uptake in human gastric adenocarcinoma SNU-1 cells, whereas resveratrol suppressed both synthesis of DNA and generation of endogenous O(2)(-) but stimulated nitric oxide (NO) synthase (NOS) activity. To address the role of NO in the antioxidant action of resveratrol, we measured the effect of sodium nitroprusside (SNP), an NO donor, on O(2)(-) generation and on [(3)H]thymidine incorporation. SNP inhibited DNA synthesis and suppressed ionomycin-stimulated O(2)(-) generation in a concentration-dependent manner. Our results revealed that the antioxidant action of resveratrol toward gastric adenocarcinoma SNU-1 cells may reside in its ability to stimulate NOS to produce low levels of NO, which, in turn, exert antioxidant action. Resveratrol-induced inhibition of SNU-1 proliferation may be partly dependent on NO formation, and we hypothesize that resveratrol exerts its antiproliferative action by interfering with the action of endogenously produced reactive oxygen. These data are supportive of the action of NO against reactive oxygen and suggest that a resveratrol-rich diet may be chemopreventive against gastric cancer.     

Fludarabine nucleoside modulates nuclear "survival and death" proteins in resistant chronic lymphocytic leukemia cells

The nuclear mechanisms by which fludarabine nucleoside (F-ara-A) induces apoptosis have been investigated in human MEC1 cells derived from B-cell chronic lymphocytic leukemia. Upon treatment of cells with F-ara-A (100 μM, 72 hours), 15 nuclear proteins changed in abundance by more than 2-fold. Nuclear proteins up-regulated included calmodulin (4.3-fold), prohibitin (3.9-fold), β-actin variant (3.7-fold), and structure-specific recognition protein 1 (3.7-fold); those down-regulated included 60S ribosomal protein P2B (0.12-fold), fumarate hydratase (0.19-fold), splicing factor arginine/serine-rich 3 (0.35-fold), and replication protein A2 (0.42-fold). These changes in the levels of specific proteins promote survival or apoptosis; because the end result is apoptosis of MEC1 cells, apoptotic effects predominate.     

Nordentatin Inhibits Neuroblastoma Cell Proliferation and Migration through Regulation of GSK-3 Pathway

Cancer is caused by abnormal cell changes leading to uncontrolled cell growth. The specific characteristics of cancer cells, including the loss of apoptotic control and the ability to migrate into and invade the surrounding tissue, result in cancer cell metastasis to other parts of the body. Therefore, the inhibition of the proliferation, migration, and invasion of cancer cells are the principal goals in the treatment of cancer. This study aimed to investigate the inhibitory activity of nordentatin, a coumarin derivative isolated from Clausena harmandiana, regarding the proliferation and migration of human neuroblastoma cells (SH-SY5Y). Nordentatin at a concentration of 100 µM showed cell cytotoxicity toward SH-SY5Y that was significantly different from that of the control group (p < 0.01) at 24, 48, and 72 h. Moreover, nordentatin inhibited SH-SY5Y proliferation by inhibiting the antiapoptotic protein Mcl-1, leading to the cleavage of caspase-3 and resulting in the inhibition of a migratory protein, MMP-9, through the GSK-3 pathway (compared with cells treated with a GSK inhibitor). These results suggest that nordentatin inhibited the proliferation and migration of neuroblastoma cells through the GSK-3 pathway.     

Anti-mitotic potential of 7-diethylamino-3(2'-benzoxazolyl)-coumarin in 5-fluorouracil-resistant human gastric cancer cell line SNU620/5-FU

In this study, we investigate an anti-mitotic potential of the novel synthetic coumarin-based compound, 7-diethylamino-3(2'-benzoxazolyl)-coumarin, in 5-fluorouracil-resistant human gastric cancer cell line SNU-620-5FU and its parental cell SNU-620. It exerts the anti-proliferative effects with similar potencies against both cancer cells, which is mediated by destabilization of microtubules and subsequent mitotic arrest. Furthermore, this compound enhances caspase-dependent apoptotic cell death via decreased expression of anti-apoptotic genes. Taken together, our data strongly support anti-mitotic potential of 7-diethylamino-3(2'-benzoxazolyl)-coumarin against drug-resistant cancer cells which will prompt us to further develop as a novel microtubule inhibitor for drug-resistant cancer chemotherapy.     

Structure-function relationship of synthetic sulfoquinovosyl-acylglycerols as mammalian DNA polymerase inhibitors

We reported previously that sulfo-glycolipids such as sulfoquinovosyl-diacylglycerol (SQDG) and sulfoquinovosyl-monoacylglycerol (SQMG) are potent inhibitors of DNA polymerase alpha and beta and antineoplastic agents. Then, we succeeded in synthesizing SQDG and SQMG chemically, including their stereoisomers, glucopyranosyl-diacylglycerol (GDG) and glucopyranosyl-monoacylglycerol (GMG). In this study, we demonstrated the structure-function relationship of the synthetic sulfo-glycolipids to DNA polymerase alpha and beta and their relationship to the cytotoxic activity. Both SQDG and SQMG inhibited the activity of mammalian DNA polymerase alpha with IC(50) values of 3-5 microM, but GMG only moderately inhibited it. GDG, diacylglycerol (DG), and monoacylglycerol (MG) did not influence any of the DNA polymerase activities. The sulfate moiety in the quinovose was important in inhibiting the enzyme activity. The one-fatty-acid-sulfo-glycolipids, SQMG, GMG, and MG, prevented the growth of NUGC-3 human gastric cancer cells and induced apoptotic cell death, but the two-fatty-acid-sulfo-glycolipids, SQDG, GDG, and DG, did not. SQMG and GMG could halt the cell cycle at the G1 phase, but the cell cycle was not changed by MG. The relationship between the DNA polymerase inhibition and the cell growth effect by these compounds are discussed.     

Gangliosides Attenuate Ethanol-Induced Apoptosis in Rat Cerebellar Granule Neurons

Ethanol significantly enhances cell death of differentiated rat cerebellar granule neurons on culture in a serum-free medium containing a depolarizing concentration of KCl (25 mM), 5 M MK-801 (an NMDA receptor antagonist), and 20–200 mM ethanol for 1–4 days. Cell death augmented by ethanol was concentration- and time-dependent with neurons displaying hallmark apoptotic morphology and DNA fragmentation that correlated with the activation of cytosolic caspase-3. Inclusion of 5 M MK-801 or 100 M glycine in culture media did not alter rates of cell death indicating ethanol toxicity is mediated via an NMDA receptor-independent pathway. Preincubation with 50 M gangliosides GM1, GD1a, GD1b or GT1b for 2 h, or preincubation with 10 M LIGA₂₀ (a semisynthetic GM1 with N-dichloroacetylsphingosine) for 10 min, attenuated caspase-3 activity and ethanol-induced cell death. Data show native gangliosides and a synthetic derivative are potently neuroprotective in this model of ethanol toxicity, and potentially serve as useful probes to further unravel the mechanisms relevant to neuronal apoptosis.     

Elimination of cells with abnormal nuclei in human epidermoid carcinoma cell line A431 by α-lipoic acid

Skin is usually exposed to adverse environmental conditions that may cause pathological cell proliferation and malignant transformation. Antioxidants are able to affect these processes and eliminate transformed cells. The purpose of this work was to investigate the effect of α-lipoic acid (ALA) on human epidermoid carcinoma cell line A431. It was found that 100, 200, 300, 500 μM ALA added for 24, 48, 72 h inhibited cell proliferation and stimulated apoptosis. Most dying cells have abnormal nuclei (micronuclei, giant nuclei, nuclei with buds). Electron microscopy showed that cells with normal nuclear phenotypes after treatment with 200 μM ALA for 48 h had ultrastructural organizations typical for control cells. Thus, α-ALA not only triggers the apoptosis of carcinoma cells, but it may also activate the mechanism for eliminating cells with abnormal numbers of chromosomes.     

EGFR and PKC are involved in the activation of ERK1/2 and p90 RSK and the subsequent proliferation of SNU-407 colon cancer cells by muscarinic acetylcholine receptors

We have previously shown that muscarinic acetylcholine receptors (mAChRs) enhance SNU-407 colon cancer cell proliferation via the ERK1/2 pathway. Here, we examined the signaling pathways linking mAChR stimulation to ERK1/2 activation and the subsequent proliferation of SNU-407 cells. The inhibition of the epidermal growth factor receptor (EGFR) by AG1478 or protein kinase C (PKC) by GF109203X significantly reduced carbachol-stimulated ERK1/2 activation and cell proliferation. Cotreatment of the cells with AG1478 and GF109203X produced an additive effect on carbachol-stimulated ERK1/2 activation, suggesting that the EGFR and PKC pathways act in parallel. The p90 ribosomal S6 kinases (RSKs) are downstream effectors of ERK1/2 and are known to have important roles in cell proliferation. In SNU-407 cells, carbachol treatment induced RSK activation in an atropine-sensitive manner, and this RSK activation was decreased by the inhibition of either EGFR or PKC. Moreover, the RSK-specific inhibitor BRD7389 almost completely blocked carbachol-stimulated cell proliferation. Together, these data indicate that EGFR and PKC are involved in mAChR-mediated activation of ERK1/2 and RSK and the subsequent proliferation of SNU-407 colon cancer cells.     

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.Abbreviations VNA Viable non-apoptotic cells - VA Viable apoptotic cells - NVNA Nonviable non-apoptotic or necrotic cells - NVA Nonviable apoptotic cells - CF Chromatin-free cells (late nonviable apoptotic cells) - AO Acridine orange - EB Ethidium Bromide - MAb Monoclocnal antibody - D.O. Dissolved oxygen - qMAb Specific MAb production rate (mg. (10⁹ cells)^–1.day^–1) - Specific growth rate (h^–1) - X_v Viable cell number (10⁵ cells.mL^–1) - X_t Total cell number (10⁵ cells.mL^–1) - Y_lac/glc Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)     

Epidermal growth factor-like growth factors prevent apoptosis of alcohol-exposed human placental cytotrophoblast cells

Maternal alcohol abuse during pregnancy can produce an array of birth defects comprising fetal alcohol syndrome. A hallmark of fetal alcohol syndrome is intrauterine growth retardation, which is associated with elevated apoptosis of placental cytotrophoblast cells. Using a human first trimester cytotrophoblast cell line, we examined the relationship between exposure to ethanol and cytotrophoblast survival, as well as the ameliorating effects of epidermal growth factor (EGF)-like growth factors produced by human cytotrophoblast cells. After exposure to 0-100 mM ethanol, cell death was quantified by the TUNEL method, and expression of the nuclear proliferation marker, Ki67, was measured by immunohistochemistry. The mode of cell death was determined by assessing annexin V binding, caspase 3 activation, pyknotic nuclear morphology, reduction of TUNEL by caspase inhibition, and cellular release of lactate dehydrogenase. Ethanol significantly reduced proliferation and increased cell death approximately 2.5-fold through the apoptotic pathway within 1-2 h of exposure to 50 mM alcohol. Exposure to 25-50 mM ethanol significantly increased transforming growth factor alpha (TGFA) and heparin-binding EGF-like growth factor (HBEGF), but not EGF or amphiregulin (AREG). When cytotrophoblasts were exposed concurrently to 100 mM ethanol and 1 nM HBEGF or TGFA, the increase in apoptosis was prevented, while EGF ameliorated at 10 nM and AREG was weakly effective. HBEGF survival-promoting activity required ligation of either of its cognate receptors, HER1 or HER4. These findings reveal the potential for ethanol to rapidly induce cytotrophoblast apoptosis. However, survival factor induction could provide cytotrophoblasts with an endogenous cytoprotective mechanism.     

Cadmium tolerance in tobacco cell culture and its relevance to temperature stress

Growth of unselected tobacco (Nicotiana tabacum W38) cell suspension cultures was reduced by 50–200 M cadmium (Cd) in the culture medium and cells were killed by 400 M Cd. Tolerance to Cd was increased either by using rapidly growing cells or by culturing cells at higher densities. Cell lines tolerant to 2 mM Cd were established by progressively elevating levels of Cd in the culture medium. The Cd tolerance was not due to differences in uptake between unselected and Cd-tolerant cell lines, and the tolerance to Cd was not lost during long term culture in the absence of Cd. Cd-tolerant cells also showed higher tolerance to heat shock (37.5°C, 2–8 hours) and cold treatments (4°C, 1–7 days) than the unselected cells.