Chromosomal instability and its relationship to other end points of genomic instability

Chromosomal destabilization is one end point of the more general phenomenon of genomic instability. We previously established that chromosomal instability can manifest in clones derived from single progenitor cells several generations after X-irradiation. To understand the potential relationship between chromosomal destabilization and the other end points of genomic instability, we generated a series of chromosomally stable and unstable clones by exposure to X-rays. All clones were derived from the human-hamster hybrid line GM10115, which contains a single copy of human chromosome 4 in a background of 20-24 hamster chromosomes. These clones were then subjected to a series of assays to determine whether chromosomal instability is associated with a general "mutator phenotype" and whether it modulates other end points of genomic instability. Thus, we analyzed clones for sister chromatid exchange, delayed reproductive cell death, delayed mutation, mismatch repair, and delayed gene amplification. Statistical analyses performed on each group of chromosomally stable and unstable clones indicated that, although individual clones within each group were significantly different from unirradiated clones for many of the end points, there was no significant correlation between chromosomal instability and sister chromatid exchange, delayed mutation, and mismatch repair. Delayed gene amplification was found to be marginally correlated to chromosomal instability (P < 0.1), and delayed reproductive cell death (the persistent reduction in plating efficiency after irradiation) was found to be significantly correlated (P < 0.05). These correlations may be explained by chromosomal destabilization, which can mediate gene amplification and can result in cellular lethality. These data implicate multiple molecular and genetic pathways leading to different manifestations of genomic instability in GM10115 cells surviving exposure to DNA-damaging agents.     

Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX

The mode of cell death induced by photodynamic treatment (PDT) was studied in two cell lines cultured in monolayer, V79 Chinese hamster fibroblasts and WiDr human colon adenocarcinoma cells. The cells were incubated with 5-aminolaevulinic acid (5-ALA) as a precursor for the endogenously synthesised protoporphyrin IX, which was activated by light. Free DNA ends, owing to internucleosomal DNA cleavage in apoptotic cells, were stained specifically with a fluorescent dye in the terminal deoxynucleotidyl transferase (TdT) assay. The free DNA ends were measured by flow cytometry and the fractions of apoptotic cells determined. Total cell death was measured in a cell survival assay to determine the necrotic fraction after subtraction of the apoptotic fraction. V79 cells did undergo apoptosis while WiDr cells were killed only through necrosis. With time, the apoptotic fraction of V79 cells increased until a maximum was reached about 3-4 h after ALA-PDT treatment. For increasing ALA-PDT doses, a maximal apoptotic fraction 75-85% of the cells was measured at about 85% of total cell death. The flow cytometric assay of apoptosis was confirmed by the typical ladder of oligonucleosomal DNA fragments obtained from agarose gel electrophoresis, by fluorescence micrographs visualising the induced free DNA ends and by electron micrographs showing the typical morphology of apoptotic cells.     

Bcl-2 protects murine erythroleukemia cells from p53-dependent and -independent radiation-induced cell death

To better understand the molecular basis of radiation-induced cell death, we studied the role of the bcl-2 oncogene and the p53 tumor suppressor gene in this process. A temperature-sensitive mutant of murine p53 (p53Val-135) and/or bcl-2 was transfected into murine erythroleukemia cells (MEL, DP16-1, which are null in p53). We demonstrate that radiation-induced cell death occurs by both p53-dependent and -independent pathways and overexpression of bcl-2 modulates both pathways. When viability was measured 24 h post-radiation, cells that had been briefly exposed to wtp53 immediately after X-ray irradiation had decreased survival as compared to unirradiated cells expressing wtp53 or X-ray irradiated DP16-1 cells. However, at later times X-ray irradiated parental DP16-1 cells also had decreased survival compared to the unirradiated control. This decrease in survival began 48 h following radiation. Bcl-2 prevented radiation-induced cell death in DP16-1 cells expressing wtp53 and delayed radiation-induced cell death in DP16-1 cells without wtp53. X-ray irradiated cells expressing wtp53 displayed microscopic and biochemical characteristics consistent with cell death due to apoptosis. DP16-1 cells which were untransfected or co-transfected with wtp53 and bcl-2 displayed characteristics of cells undergoing necrosis. These results suggest that radiation-induced cell death occurs by both p53-dependent and p53-independent pathways. The p53-dependent pathway results in cell death via apoptosis and occurs approximately 24 h following radiation. The p53-independent pathway does not appear to involve apoptosis and occurs at a later time, starting 48 h after X-ray exposure. Thus, bcl-2 protects cells from p53-dependent radiation-induced apoptotic cell death and attenuates p53-independent radiation-induced cell death.     

Involvement of p85 in p53-dependent apoptotic response to oxidative stress

Reactive oxygen species have damaging effects on cellular components and so trigger defensive responses by the cell and even programmed cell death, although the mechanisms by which mammalian cells transmit signals in response to oxidative damage are unknown. We report here that the protein p85, a regulator of the signalling protein phosphatidyl-3-OH kinase (PI(3)K), participates in the cell death process that is induced in response to oxidative stress and that this role of p85 in apoptosis does not involve PI(3)K. We show that disruption of p85 by homologous recombination impairs the cellular apoptotic response to oxidative stress. Because the protein p53 is required for cell death induced by oxidative damage, we examined the relation between p85 and p53. Using a chimaeric p53 fusion protein with the oestrogen receptor (p53ER) to supply p53 (p53 is induced upon binding of p53ER to oestradiol) in a p53-deficient cell line, we found that p85 is upregulated by p53 and that its involvement in p53-mediated apoptosis is independent of PI(3)K. We propose that p85 acts as a signal transducer in the cellular response to oxidative stress, mediating cell death regulated by p53.     

Survey of nurse anesthesia practice, education, and regulation in 96 countries

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21WAF/CIP1 protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.     

Role of p53 in UVB-induced apoptosis in human HaCaT keratinocytes

Apoptosis represents an active form of cell death that is involved in the control of tissue homeostasis and in the deletion of DNA-damaged cells. Because the product of the tumor suppressor gene p53 has been demonstrated to be crucial for the induction of apoptosis in certain cell types, the present study was aimed at elucidating its role in ultraviolet-induced apoptosis in HaCaT keratinocytes. After in vitro ultraviolet B irradiation, p53 protein levels were noted to increase prior to the induction of apoptosis in a time- and concentration-dependent fashion. This increase could not be inhibited by the protein synthesis inhibitor cycloheximide. Because HaCaT keratinocytes are known to bear two p53 point mutations and because it is unclear whether p53 in HaCaT cells is still functional regarding induction of apoptosis, HaCaT cells were stably transfected with wild-type p53 cDNA inserted into the expression vector pCMV-Neo-Bam in sense (pC53-SN3) and anti-sense (pC53-ASN) direction. After selection with geniticin, growing colonies were screened for the presence of the transfected cDNA constructs by polymerase chain reaction. Cell clones bearing the anti-sense product were further analyzed for p53 expression by western blotting. Clones showing reduced p53 protein levels were irradiated with ultraviolet B light, and there was a clear reduction of apoptosis in the pC53-ASN bearing cell clones compared with the parental HaCaT cells. These studies demonstrate that blocking mutated p53 can partially block apoptosis in HaCaT keratinocytes and furthermore can confirm the key role for p53 in ultraviolet-induced apoptosis in human keratinocytes. Moreover, HaCaT keratinocytes and their p53-transfectants provide a convenient model that allows for further detailed analyses of apoptosis-associated biochemical and molecular events in human keratinocytes.     

Tumor necrosis factor-induced apoptosis stimulates p53 accumulation and p21WAF1 proteolysis in ME-180 cells

Tumor necrosis factor (TNF)-mediated apoptotic signaling has been characterized by activation of specific protease or protein kinase cascades that regulate the onset of apoptosis. TNF has also been shown to induce oxidative or genotoxic stress in some cell types, and apoptotic potential may be determined by the cellular response to this stress. To determine the role of genotoxic stress in TNF-mediated apoptosis, we examined cellular accumulation of p53 in TNF-treated ME-180 cells selected for apoptotic sensitivity (ME-180S) or resistance (ME-180R) to TNF. Although TNF was able to activate receptor-mediated signaling in either cell line, p53 accumulation was measurable only in apoptotically sensitive ME-180S cells. TNF-induced changes in p53 levels were detected 1 h after treatment, and peak levels were measurable 4-8 h after TNF exposure. TNF was unable to induce p21WAF1 in either cell line but affected the stability of this protein in apoptotically responsive ME-180S cells. Evidence of p21WAF1 proteolysis was detected by monitoring the appearance of a 16-kDa immunoblottable p21WAF1 fragment, which became detectable 4 h after TNF addition and increased in content before the onset of DNA fragmentation (16-24 h). The kinetics of p21WAF1 proteolysis closely paralleled those of poly(ADP-ribose) polymerase, suggesting cleavage of p21WAF1 by activation of an apoptotic protease. Pretreatment of ME-180S cells with the apoptotic protease inhibitor YVAD blocked TNF-induced apoptosis and prevented both poly(ADP-ribose) polymerase and p21WAF1 degradation but did not affect p53 induction. These results provide evidence for the early onset of genotoxic stress in cells committed to TNF-mediated apoptosis and for divergence in propagation of this signal in non-responsive cells. In addition, TNF-induced p21WAF1 proteolysis may be mediated by an apoptotic protease and may contribute to the apoptotic process by disrupting p53 signaling, altering cell cycle inhibition, and limiting cellular recovery from genotoxic stress.     

Increased apoptosis and necrosis of coronary plaques in unstable angina

In acute coronary syndromes, arteriosclerotic plaques are characterized by inflammation and decreased smooth muscle cell density. The underlying pathogenic processes remain unclear. Among others, increased programmed cell death (apoptosis) is postulated. Coronary atherectomy specimens from 26 patients with unstable angina (group 1) and from 24 patients with stable angina (group 2) were examined, using immunohistochemistry (TUNEL test to detect fragmented DNA) and transmission electron microscopy. The objectives of the present study were to evaluate plaque group differences in the cellular composition, to detect and quantify cell death, and to differentiate between apoptosis and necrosis. Group 1 lesions contained more macrophages and lymphocytes as well as significantly (p = 0.01) less smooth muscle cells compared with group 2 lesions, whereas both revealed a comparable cell density. All plaques showed signals for fragmented DNA. TUNEL-positive cells were seen more frequently in lesions with unstable angina (p = 0.04). Ultrastructural analysis revealed signs of programmed cell death, such as nuclear alterations, cellular condensation due to lost adhesion, and apoptotic bodies. Importantly, group I lesions comprised significantly more apoptotic SMCs and apoptotic macrophages compared with group 2 lesions (28% vs. 16%; p = 0.02). Also, cellular necroses were found to be increased in lesions with unstable angina (18% vs. 8%; p = 0.02). The density of macrophages showed a positive correlation to the incidence of cellular necroses in group 1 lesions (r = 0.44; p = 0.02), but not in group 2 lesions. In both plaque groups, this determinant was independent from cellular apoptosis, also at high levels as found with unstable angina. The present study on coronary atherectomy specimens with unstable angina reveals intimal macrophage infiltration and the density of apoptotic as well as necrotic intimal cells to be increased, whereas the content of intact SMCs was reduced. Increased, macrophage-independent apoptosis strongly points to the presence of one or several pro-apoptotic intimal factor(s) predisposing to plaque rupture. Implications of our findings may be directed to identify this (these) factor(s) and to modulate endogenous apoptotic activity with the ultimate goal to raise regional smooth muscle cell density.     

Apoptosis in histiocytic necrotizing lymphadenitis

Cell death can now be divided into necrosis and apoptosis, which are different in their morphology, biochemistry and biological significance. The present study was designed to investigate cell death in histiocytic necrotizing lymphadenitis (HNL). The features of cell death in 10 cases of HNL were analyzed using histiomorphology, ultrastructure and in situ apoptosis detection (ApopTag) methods. Two patterns of cell death were discerned. One was apoptosis of individual cells and the other was necrosis. The first pattern could be observed in all cases and the morphological features of the dead cells were consistent with those of apoptosis, which included distinctive cell volume shrinking and chromatin condensation. The apoptotic cells and bodies could frequently be found to be phagocytosed by the histiocytes. ApopTag was positively stained in most of the morphologically apoptotic cells. By double staining, most ApopTag positive cells were found to be T lymphocytes. A previous report showed that the majority of the proliferative cells were T lymphocytes. Based on those results, if was speculated that the main pathological characteristics of HNL therefore consisted of apoptosis and the proliferation of T lymphocytes.     

E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells

Integrin-basement membrane interactions provide essential signals that promote survival and growth of epithelial cells, whereas loss of such adhesions triggers programmed cell death. We found that HSC-3 human squamous carcinoma cells survived and grew readily as monolayers, but when they were suspended as single cells, they ceased proliferating and entered into the apoptotic death pathway, characterized by DNA fragmentation. In contrast, if the suspended carcinoma cells were permitted to form E-cadherin-mediated multicellular aggregates, they not only survived but proliferated. However, aggregated normal keratinocytes were unable to survive in suspension culture and rapidly became apoptotic. Anchorage independence and resistance to apoptosis of HSC-3 cell aggregates required high levels of extracellular Ca2+ and was inhibited with function-perturbing anti-E-cadherin antibody. Resistance to suspension-induced apoptosis in cell aggregates paralleled the up-regulation of Bcl-2 but occurred in the absence of focal adhesion kinase activation. Analysis of suspension-induced death in a set of cloned squamous epithelial cell lines with different levels of E-cadherin expression revealed that receptor-positive cell clones evaded apoptosis and proliferated in three-dimensional aggregate culture, whereas cadherin-negative clones failed to survive. Collectively, these observations indicate that cadherin-mediated intercellular adhesions generate a compensatory mechanism that promotes anchorage-independent growth and suppresses apoptosis.     

p53-deficient mice are protected against adrenalectomy-induced apoptosis

The p53 tumor suppressor gene, an important regulator of the cell cycle, has been implicated in apoptotic cell death in vitro, and more recently in neuronal degeneration in vivo. The present study investigated the importance of p53 expression in the apoptotic death of hippocampal granule cells following adrenalectomy. Mice, either homozygous or heterozygous for the p53 null allele and wild-type controls were sacrificed 16 days after adrenalectomy. Hippocampal morphology was assessed in paraffin sections stained with hematoxylin and eosin. Cells exhibiting features characteristic of apoptosis were evident in hippocampi from wild-type mice. A significant decrease in the number of apoptotic cells was observed in both homozygous and heterozygous mice. These findings demonstrate that absence or attenuation of p53 expression protects granule cells from adrenalectomy-induced apoptosis and, combined with the results of other studies, suggest that p53 is required for certain types of neuronal degeneration.     

Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1

The ability of beta cells to endure assaults by various environmental agents, including toxins and viruses, may be relevant to the development of diabetes. We have examined the mode of cell death caused by streptozotocin (STZ) in a murine pancreatic beta cell line, INS-1. Apoptosis was identified by detection of initial endonuclease-mediated DNA strand breaks by DNA gel electrophoresis. Apoptosis and necrosis were distinguished morphologically by light and electron microscopy. Higher rates of apoptosis, as compared to necrosis, were observed when cells were exposed to 15 mM STZ for 1 hr followed by a 24 hrs recovery period. Higher doses of STZ (30 mM) caused the cells to undergo necrosis (22%) as well as apoptosis (17%). These results suggest that the cytotoxic effect of STZ, at low doses, on beta cells involves the activation of the apoptotic pathway, whereas, at high doses, the mode of beta cell death is predominantly necrosis.     

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.     

Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.     

Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats

Programmed cell death in the myocardium has been linked to ischemia reperfusion injury as well as to excessive mechanical forces associated with increases in ventricular loading. Moreover, hypoxia activates the suicide program of cardiac myocytes in vitro. Because the supplied portion of the ventricular wall is ischemic and subjected to high levels of systolic and diastolic stresses (acutely after coronary artery occlusion), apoptosis and necrosis may contribute independently to myocyte cell death after infarction. Therefore, myocardial infarction was produced in rats, and, after the determination of ventricular hemodynamics, the contribution of apoptotic and/or necrotic myocyte cell death to infarct size was measured quantitatively from 20 minutes to 7 days after coronary artery occlusion. Programmed cell death was assessed by the terminal deoxynucleotidyl transferase assay and by the electrophoretic detection of DNA laddering. Myocyte necrosis was evaluated by myosin monoclonal Ab labeling. Moreover, the expression of Bcl-2, Bax, and Fas proteins in myocytes was examined by immunocytochemistry. Myocyte cell death by apoptosis and necrosis comprised nearly 3 million myocytes at 2 hours. Apoptotic cell death involved 2.8 million cells and necrotic cell death only 90,000 myocytes. Apoptosis continued to represent the major independent form of myocyte cell death, affecting 6.6 million myocytes at 4.5 hours. Myocyte necrosis peaked at 1 day, including 1.1 million myocytes. DNA electrophoretic analysis confirmed these observations by showing nucleosomal ladders at 2-3 hours, 4.5 hours, 1 day, and 2 days after coronary artery occlusion. Myocytes showing both DNA strand breaks and myosin labeling were a prominent aspect of myocardial damage only after 6 hours. Finally, the expression of Bcl-2 and Fas in myocytes increased 18-fold and 131-fold, respectively. In conclusion, programmed myocyte cell death is the major form of myocardial damage produced by occlusion of a major epicardial coronary artery, whereas necrotic myocyte cell death follows apoptosis and contributes to the progressive loss of cells with time after infarction. The enhanced expression of Fas may be implicated in the activation of apoptosis in spite of the increase in Bcl-2, which tends to preserve cell survival.     

Inhibition of radiation-induced G2 delay potentiates cell death by apoptosis and/or the induction of giant cells in colorectal tumor cells with disrupted p53 function

We have previously identified a p53-independent apoptotic response that is delayed until 48-72 h after irradiation of colorectal adenoma and carcinoma cells. Because the delay appears to be in part due to a transient G2 cell cycle arrest, the importance of this checkpoint in the mechanism of ionizing radiation (IR)-induced death of colorectal tumor cells was investigated. An adenoma cell line with (282Arg-->Trp) mutant p53 (S/RG/C2) and a carcinoma cell line (PC/JW/FI) lacking p53 protein treated with 5 Gy IR in the presence of 1.5 mm caffeine (CAF) reduced IR-induced G2 arrest and increased the level of apoptosis (1.5-1.6-fold) 24 h after treatment. Increased IR apoptotic cell death with CAF significantly reduced IR cell survival over a 7-day period in S/RG/C2 and PC/JW/FI. To investigate whether CAF radiosensitization correlated with lack of wild-type (wt) p53, we studied transfected derivatives of an adenoma-derived cell line (PC/AA/C1), in which the endogenous wt p53 activity was disrupted by the expression of a dominant negative (273Arg-->His) p53 mutant protein (designated AA/273p53/B). This p53-defective cell line was also radiosensitized by CAF, whereas the vector control (AA/PCMV/D), which retained wt p53 activity, was not. In addition, as with the S/RG/C2 and PC/JW/FI cell lines, the 7-day IR cell survival was reduced significantly in AA/273p53/B compared with the vector control cell line. This suggests that radiosensitization by CAF and increased cell death is dependent on loss of wt p53 function. Interestingly, radiosensitization of the AA/273p53/B cell line was not associated with accelerated apoptosis but correlated with increased polyploid giant cells, which have been associated with disruption of cell cycle checkpoints and genomic instability. These results demonstrate that G2 checkpoint inhibition with CAF leads to preferential IR cell killing in cell lines in which wt p53 is inactivated and that this increased cell killing is not necessarily dependent on increased IR-induced apoptosis.     

Correlates of well-being in mothers of children and adolescents with cystic fibrosis

The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a biochemical basis, genistein is a competitive inhibitor of tyrosine kinases and the DNA synthesis-related enzyme, topoisomerase-II (topo-II). Exposure of mammalian cells to genistein results in DNA damage that is similar to that induced by the topo-II inhibitor and chromosomal mutagen, m-amsa. In order to determine the potential genotoxicity of genistein, human lymphoblastoid cells which differ in the functional status of the tumor suppressor gene, p53, were exposed to genistein and the induction of micronuclei quantified by microscopic analysis. In addition, the mutant fraction at the thymidine kinase (tk) locus (both the normal-growth and slow-growth phenotypes) was determined by resistance to trifluorothymidine (TFT) and at the hypoxanthine phosphoribosyl transferase (hprt) locus by resistance to 6-thioguanine (6-TG). Flow cytometric analysis of the percentage of viable, apoptotic and degenerating cells was utilized to determine the rate and kinetics of cell death after genistein exposure. The detection of micronuclei in both cell lines indicated that genistein-induced damage had occurred in both AHH-1 tk+/- and L3. Linear regression analysis detected a significant increase in the number of 6-TG-resistant clones in both AHH-1 tk+/- (p53+/-) and L3 (p53+/+). A comparison of slopes revealed no difference between the lines. In contrast, a significant, concentration-dependent increase in the number of TFT-resistant clones with the slow-growth phenotype was detected in AHH-1 tk+/- (mutant p53), but not in L3 (wild-type p53). Cell death occurred primarily by apoptosis in both cell lines; however, a concentration-dependent decrease in the percentage of viable cells was detected immediately after exposure in L3, but not until 32 h after exposure in AHH-1 tk+/-. A comparison of the slopes of the concentration-response curves for the percentage of viable cells revealed no difference between the cell lines in the effect of genistein on cell viability. Our results may be interpreted that genistein is a chromosomal mutagen and that p53 functional status affects the recovery of chromosomal mutants, possibly by signalling cells into the apoptosis pathways.     

Apoptosis, cancer and cancer therapy

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis. Apoptosis is essential in the homeostasis of normal tissues of the body, especially those of the gastrointestinal tract, immune system and skin. There is increasing evidence that the processes of neoplastic transformation, progression and metastasis involve alterations in the normal apoptotic pathways. Furthermore, the majority of chemotherapeutic agents as well as radiation utilize the apoptotic pathway to induce cancer cell death. Resistance to standard chemotherapies also seems to be determined by alterations in the apoptotic pathways of cancer cells. Therefore, understanding the signals of apoptosis and the mechanism of apoptosis may allow the development of better chemo- or radiotherapeutic regimens for the treatment of cancer. Finally, components of the apoptotic pathway may represent potential therapeutic targets using gene therapy techniques.     

Evaluation of several dosing regimens of cefepime, with various simulations of renal function, against clinical isolates of Pseudomonas aeruginosa in a pharmacodynamic infection model

Development of the eye requires complex interactions between tissues, extracellular matrix and growth factors. Most cells of the optic primordia grow and differentiate into discrete ocular structures; however, other cells have death as their developmental fate. The most common mechanisms of cell death are apoptosis and necrosis. We have identified the cell death that occurs during ocular morphogenesis in ZRDCT-N mice as apoptosis. Mouse embryos, ages E8.5-E11.5, were embedded in paraffin, sectioned at 5 microns and stained with hematoxylin or by the terminal deoxytransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The spatial and temporal distribution of apoptotic cells was mapped at 0.5 day intervals using a computerized image analysis system, and 3-D reconstructions were made at each embryonic age. Our data indicate that apoptosis plays a role in normal ocular morphogenesis and provides the groundwork for studies of abnormal ocular development.