Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

The role of heritable, population-wide cell damage in neoplastic development was studied in the 28 L subline of NIH 3T3 cells. These cells differ from the 17(3c) subline used previously for such studies in their lower frequency of "spontaneous" transformation at high population density and their greater capacity to produce large, dense transformed foci. Three cultures of the 28 L subline of NIH 3T3 cells were held under the constraint of confluence for 5 wk (5 wk 1 degree assay) and then assayed twice in succession (2 degrees and 3 degrees assays) for transformed foci and saturation density. After the 2 degrees assay, the cells were also passaged at low density to determine their exponential growth rates and cloned to determine the size and morphological features of the colonies. Concurrent measurements were made in each case with control cells that had been kept only in frequent low-density passages and cells that had been kept at confluence for only 2 wk (2 wk 1 degree). Two of the three cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures produced light transformed foci, and the third produced dense foci. The light focus-forming cultures grew to twice the control saturation density in their 2 degrees assay and 6-8 times the control density in the 3 degrees assay; saturation densities for the dense focus formers were about 10 times the control values in both assays. All three of the cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures multiplied at lower rates than controls at low densities, but the dense focus formers multiplied faster than the light focus formers. The reduced rates of multiplication of the light focus formers persisted for > 50 generations of exponential multiplication at low densities. Isolated colonies formed from single cells of the light focus formers were of a lower population density than controls; colonies formed by the dense focus formers were slightly denser than the controls but occupied only half the area. A much higher proportion of the colonies from the 5 wk 1 degree cultures than the controls consisted of giant cells or mixtures of giant and normal-appearing cells. The results reinforce the previous conclusion that the early increases in saturation density and light focus formation are associated with, and perhaps caused by, heritable, population-wide damage to cells that is essentially epigenetic in nature. The more advanced transformation characterized by large increases in saturation density and dense focus formation could have originated from rare genetic changes, such as chromosome rearrangements, known to occur at an elevated frequency in cells destabilized by antecedent cellular damage.

Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.

N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-). This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases. Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features. Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine. In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase. Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage.

Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes

Streptozotocin (STZ), a glucose analogue known to induce diabetes in experimental animals, causes DNA strand breaks and subsequent activation of poly(ADPribose) polymerase (Parp). Because Parp uses NAD as a substrate, extensive DNA damage will result in reduction of cellular NAD level. In fact, STZ induces NAD depletion and cell death in isolated pancreatic islets in vitro. Activation of Parp therefore is thought to play an important role in STZ-induced diabetes. In the present study, we established Parp-deficient (Parp−/−) mice by disrupting Parp exon 1 by using the homologous recombination technique. These mice were used to examine the possible involvement of Parp in STZ-induced β-cell damage in vivo. The wild-type (Parp+/+) mice showed significant increases in blood glucose concentration from 129 mg/dl to 218, 370, 477, and 452 mg/dl on experimental days 1, 7, 21, and 60, respectively, after a single injection of 180 mg STZ/kg body weight. In contrast, the concentration of blood glucose in Parp−/− mice remained normal up to day 7, slightly increased on day 21, but returned to normal levels on day 60. STZ injection caused extensive necrosis in the islets of Parp+/+ mice on day 1, with subsequent progressive islet atrophy and loss of functional β cells from day 7. In contrast, the extent of islet β-cell death and dysfunction was markedly less in Parp−/− mice. Our findings clearly implicate Parp activation in islet β-cell damage and glucose intolerance induced by STZ in vivo.

Change in Apoplastic Aluminum during the Initial Growth Response to Aluminum by Roots of a Tolerant Maize Variety1

Root elongation, hematoxylin staining, and changes in the ultrastructure of root-tip cells of an Al-tolerant maize variety (Zea mays L. C 525 M) exposed to nutrient solutions with 20 μm Al (2.1 μm Al3+ activity) for 0, 4, and 24 h were investigated in relation to the subcellular distribution of Al using scanning transmission electron microscopy and energy-dispersive x-ray microanalysis on samples fixed by different methods. Inhibition of root-elongation rates, hematoxylin staining, cell wall thickening, and disturbance of the distribution of pyroantimoniate-stainable cations, mainly Ca, was observed only after 4 and not after 24 h of exposure to Al. The occurrence of these transient, toxic Al effects on root elongation and in cell walls was accompanied by the presence of solid Al-P deposits in the walls. Whereas no Al was detectable in cell walls after 24 h, an increase of vacuolar Al was observed after 4 h of exposure. After 24 h, a higher amount of electron-dense deposits containing Al and P or Si was observed in the vacuoles. These results indicate that in this tropical maize variety, tolerance mechanisms that cause a change in apoplastic Al must be active. Our data support the hypothesis that in Al-tolerant plants, Al can rapidly cross the plasma membrane; these data clearly contradict the former conclusions that Al mainly accumulates in the apoplast and enters the symplast only after severe cell damage has occurred.

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate

A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20–50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9–12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD/D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD/D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of confluence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.

DNA damage and p53-mediated cell cycle arrest: A reevaluation

Most mammalian cells exhibit transient delays in the G1 and G2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G1, which provides time for DNA repair. Recently, a role of p53 in the G2/M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G1 arrest, they do not subsequently arrest in G2. Moreover, wild-type p53 cells irradiated past the G1 checkpoint arrest in G2 but do not delay in the subsequent G1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.

Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development

We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80−/− embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80−/− mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80−/− mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80−/− mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair.

Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

GADD45 induction of a G2/M cell cycle checkpoint

G1/S and G2/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G2/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G2/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G2/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li–Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G2/M checkpoint is at least in part through modulation of the activity of the G2-specific kinase, cyclin B1/p34cdc2. Genetic and physiological evidence of a Gadd45-mediated G2/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G2/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 −/−) also retained a G2/M checkpoint initiated by ionizing radiation and failed to arrest at G2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G2/M checkpoints in response to specific types of DNA damage.

A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.

In vivo evidence that erythropoietin protects neurons from ischemic damage

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-d-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.