Unusual radiographic appearance of drug-induced pharyngeal angioedema and differential considerations.

A 59-year-old woman treated chronically with enalapril, an angiotensin-converting enzyme inhibitor (ACE-I) presented with difficult swallowing and speaking. Although her symptoms were clinically consistent with an adverse angioedema reaction to the ACE-I, initial imaging was not entirely consistent with our conceptual understanding of angioedema. This case report will discuss the myriad possible imaging presentations of this disease, as well as the differential diagnosis for this atypical manifestation of ACE-I-induced angioedema.

Exposure to low-level chemicals and ionizing radiation: reactive oxygen species and cellular pathways.

Reactive oxygen species (ROS), which contribute to the energy landscapes in and around cells, play numerous roles in maintaining normal cell homeostasis as components of signaling pathways. Excessively high levels of ROS, on the other hand, can lead to pronounced DNA damage and a variety of cellular responses, including cell cycle arrests, senescence, apoptosis and possibly cancer. Far less is known, however, about how supra-basal levels of ROS that can be generated in response to low doses of ionizing radiation or chemicals in the environment may bring about untoward or perhaps even beneficial cellular responses. Even so, some evidence suggests that adaptive responses that have been associated with ROS-generating stimuli can have protective effects by fundamentally altering subsequent cellular dose-response profiles to otherwise detrimental stresses. Yet, even these seemingly favorable 'adaptive' effects may have longer-term untoward consequences. Other effects that have been associated with supra-basal levels of ROS, such as enhanced states of cell proliferation, potentially could have a protective function. But again, such increases in cell growth, which may be accompanied by greater than normal ROS-mediated damage to DNA, as well may ultimately favour the expansion of cells with heritable mutations. Unfortunately, the state of the art of our current understanding of how elevated but still low-level increases in ROS that may be induced by environmental stimuli presently precluded incorporation of supra-basal ROS-associated mechanisms in predictive risk assessment models, both at the population level and at the level of individualized risk assessment.

Structure-based design of a bispecific receptor mimic that inhibits T cell responses to a superantigen.

Key surface proteins of pathogens and their toxins bind to the host cell receptors in a manner that is quite different from the way the natural ligands bind to the same receptors and direct normal cellular responses. Here we describe a novel strategy for "non-antibody-based" pathogen countermeasure by targeting the very same "alternative mode of host receptor binding" that the pathogen proteins exploit to cause infection and disease. We have chosen the Staphylococcus enterotoxin B (SEB) superantigen as a model pathogen protein to illustrate the principle and application of our strategy. SEB bypasses the normal route of antigen processing by binding as an intact protein to the complex formed by the MHC class II receptor on the antigen-presenting cell and the T cell receptor. This alternative mode of binding causes massive IL-2 release and T cell proliferation. A normally processed antigen requires all the domains of the receptor complex for its binding, whereas SEB requires only the alpha1 subunit (DRalpha) of the MHC class II receptor and the variable beta subunit (TCRVbeta) of the T cell receptor. This prompted us to design a bispecific chimera, DRalpha-linker-TCRVbeta, that acts as a receptor mimic and prevents the interaction of SEB with its host cell receptors. We have adopted (GSTAPPA)(2) as the linker sequence because it supports synergistic binding of DRalpha and TCRVbeta to SEB and thereby makes DRalpha-(GSTAPPA)(2)-TCRVbeta as effective an SEB binder as the native MHC class II-T cell receptor complex. Finally, we show that DRalpha-(GSTAPPA)(2)-TCRVbeta inhibits SEB-induced IL-2 release and T cell proliferation at nanomolar concentrations.

Inhibition of normal human lung fibroblast growth by beryllium.

Inhalation of particulate beryllium (Be) and its compounds causes chronic Be disease (CBD) in a relatively small subset ( approximately 1-6%) of exposed individuals. Hallmarks of this pulmonary disease include increases in several cell types, including lung fibroblasts, that contribute to the fibrotic component of the disorder. In this regard, enhancements in cell proliferation appear to play a fundamental role in CBD development and progression. Paradoxically, however, some existing evidence suggests that Be actually has antiproliferative effects. In order to gain further information about the effects of Be on cell growth, we: (1) assessed cell proliferation and cell cycle effects of low concentrations of Be in normal human diploid fibroblasts, and (2) investigated the molecular pathway(s) by which the cell cycle disturbing effects of Be may be mediated. Treatment of human lung and skin fibroblasts with Be added in the soluble form of BeSO(4) (0.1-100 microM) caused inhibitions of their growth in culture in a concentration-dependent manner. Such growth inhibition was found to persist, even after cells were further cultured in Be(2+)-free medium. Flow cytometric analyses of cellular DNA labeled with the DNA-binding fluorochrome DAPI revealed that Be causes a G(0)-G(1)/pre-S phase arrest. Western blot analyses indicated that the Be-induced G(0)-G(1)/pre-S phase arrest involves elevations in TP53 (p53) and the cyclin-dependent kinase inhibitor CDKN1A (p21(Waf-1,Cip1)). That Be at low concentrations inhibits the growth of normal human fibroblasts suggests the possibility of the existence of abnormal cell cycle inhibitory responses to Be in individuals who are sensitive to the metal and ultimately develop CBD.

Factors underlying the cell growth-related bystander responses to alpha particles.

Increases in cell proliferation are widely viewed as being of importance in carcinogenesis. We report that exposure of normal human lung fibroblasts to a low dose of alpha particles like those emitted by radon/radon progeny stimulates their proliferation in vitro, and this response also occurs when unirradiated cells are treated with supernatants from alpha-irradiated cells. We attribute the promitogenic response to superoxide dismutase- and catalase-inhibitable a particle-induced increases in the concentrations of transforming growth factor beta1 (TGF-beta1) in cell supernatants. TGF-beta1 at concentrations commensurate with those in the supernatants capably induces increases in intracellular reactive oxygen species (ROS) in unirradiated cells. Furthermore, the addition of supernatants from alpha-irradiated cells to unirradiated cells decreases cellular levels of TP53 and CDKN1A and increases CDC2 and proliferating cell nuclear antigen in the latter. Like the increased intracellular ROS bystander effect, this "decreased TP53/CDKN1A response" can be mimicked in otherwise untreated cells by the addition of low concentrations of TGF-beta1. Our results indicate that alpha particle-associated increases in cell growth correlate with intracellular increases in ROS along with decreases in TP53 and CDKN1A, and that these cellular responses are mechanistically coupled. As well, the proliferating cell nuclear antigen and CDC2 increases that occur along with the decreased TP53/CDKN1A bystander effect also would expectedly favor enhanced cell growth. Such processes may account for cell hyperplastic responses in the conducting airways of the lower respiratory track that occur after inhalation exposure to radon/ radon progeny, as well as, perhaps, other ROS-associated environmental stresses.

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres.

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake. Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.

DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes.

Recent findings intriguingly place DNA double-strand break repair proteins at chromosome ends in yeast, where they help maintain normal telomere length and structure. In the present study, an essential telomere function, the ability to cap and thereby protect chromosomes from end-to-end fusions, was assessed in repair-deficient mouse cell lines. By using fluorescence in situ hybridization with a probe to telomeric DNA, spontaneously occurring chromosome aberrations were examined for telomere signal at the points of fusion, a clear indication of impaired end-capping. Telomeric fusions were not observed in any of the repair-proficient controls and occurred only rarely in a p53 null mutant. In striking contrast, chromosomal end fusions that retained telomeric sequence were observed in nontransformed DNA-PK(cs)-deficient cells, where they were a major source of chromosomal instability. Metacentric chromosomes created by telomeric fusion became even more abundant in these cells after spontaneous immortalization. Restoration of repair proficiency through transfection with a functional cDNA copy of the human DNA-PK(cs) gene reduced the number of fusions compared with a negative transfection control. Virally transformed cells derived from Ku70 and Ku80 knockout mice also displayed end-to-end fusions. These studies demonstrate that DNA double-strand break repair genes play a dual role in maintaining chromosomal stability in mammalian cells, the known role in repairing incidental DNA damage, as well as a new protective role in telomeric end-capping.

Enhanced immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells analyzed by phase-sensitive flow cytometry.

BACKGROUND: The primary source of interference in immunofluorescence measurements by flow cytometry is background autofluorescence. METHODS: Using human lung fibroblasts (HLFs) as an autofluorescent cell model, unfixed HLFs and HLFs fixed in methanol, ethanol, formaldehyde, paraformaldehyde and glutaraldehyde were analyzed by phase-sensitive flow cytometry to compare their fluorescence intensity and lifetime histograms. Based on these results, a surface antigen on HLFs was labeled with a fluorescein isothiocyanate (FITC) conjugated antibody and fixed in glutaraldehyde, and the cells were analyzed by conventional and phase-resolved methods. RESULTS: The lifetimes of unfixed and ethanol-, methanol-, paraformaldehyde- and formaldehyde-fixed HLFs were in the 1.7-1.9 nanosecond (ns) range, with coefficients of variation 25-35%. Since the autofluorescence lifetime histograms of unfixed and fixed HLFs partially overlapped the 3.5 ns lifetime histogram of FITC-labeled microspheres, which were used to approximate FITC-antibody labeling of HLFs, the ability to resolve FITC-labeled probe, based on differences in the FITC and autofluorescence lifetimes, was severely limited. When HLFs labeled with an FITC-antibody cell-surface marker were fixed in glutaraldehyde (autofluorescence lifetime 0.9-1.4 ns, coefficient of variation approximately 11%) and analyzed by phase-resolved methods, the results showed that FITC-antibody labeling could be readily resolved from background autofluorescence. CONCLUSIONS: Phase-sensitive detection improves the immunofluorescence measurement resolution of surface antigens on highly autofluorescent, glutaraldehyde-fixed cells. Cytometry 37: 275-283, 1999. Published 1999 Wiley-Liss, Inc.

Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry.

We report a flow cytometric fluorescence lifetime-based method to discriminate damaged/dead from viable cells in immunofluorescently labeled populations using propidium iodide as a dye-exclusion viability probe. Fluorescence signals from propidium iodide and the anti-thymus cell-surface immunofluorescence marker fluorochromes, phycoerythrin and phycoerythrin/Texas Red (tandem conjugate), which have overlapping emission spectra with propidium iodide, are resolved based on differences in their fluorescence emission lifetimes using phase-sensitive detection. Mouse thymus cell samples were first labeled separately with anti-Thy 1.2 antibody directly conjugated to phycoerythrin and to phycoerythrin/Texas Red and propidium iodide. Labeled cells were then analyzed to determine the lifetimes of the immunofluorescence markers and propidium iodide. Based on these results, rat and mouse thymocytes labeled with anti-Thy 1.1 conjugated to phycoerythrin and anti-Thy 1.2 conjugated to phycoerythrin/Texas Red, respectively, were suspended in phosphate buffered saline containing propidium iodide, and were analyzed as they passed through a flow chamber and crossed a high-frequency, intensity-modulated (sinusoidal) laser excitation beam. The resulting immunofluorescence and propidium iodide signals were resolved based on differences in fluorescence lifetimes expressed as phase shifts using phase-sensitive detection and displayed as frequency distribution histograms and bivariate contour diagrams. This technology provides a new method to resolve immunofluorescence and propidium iodide signals from overlapping fluorescence emission spectra and a flow cytometric lifetime-based technique to quantify damaged/dead cells in immunofluorescence studies.

Alpha particles induce the production of interleukin-8 by human cells.

The pulmonary microenvironment is a primary target for alpha particles like those emitted by inhaled radon and its progeny. While exposure to alpha particles has recently been associated with the generation of extracellular and intracellular reactive oxygen species (ROS; Cancer Res. 57, 3963-3971, 1997), little is known about how exposure to alpha particles may affect the generation of oxidative stress-related mediators in the respiratory tract. Interleukin-8 (IL8) is a cytokine recognized for its potent role as a chemoattractant and activator of polymorphonuclear leukocytes. Oxidative stress can up-regulate expression of the gene that encodes IL8 (IL8) in a variety of cell types. In this study, we set out to investigate a potential linkage between the generation of ROS and production of IL8 in alpha-particle-irradiated normal human lung fibroblasts. ELISA revealed that exposure of the fibroblasts to low doses of alpha particles (3.6-19 cGy) caused significant increases in generation of the IL8 protein as early as 30 min after irradiation. Northern blot analyses revealed that such increases were associated with increased IL8 mRNA levels. Cells exposed to alpha particles in the presence of antioxidants, i.e. superoxide dismutase and dimethyl sulfoxide, resulted in significant decreases in extracellular IL8 protein levels. Similar results were obtained with cells treated with dexamethasone, an inhibitor of transcription. Our results indicate that alpha-particle-induced increases in production of IL8 occur temporally in parallel with elevated production of ROS. Conceivably, such production of IL8 induced by alpha particles may contribute to an inflammatory response in the lower respiratory tract. Additionally, the promitogenic effects of IL8 may be a factor in hyperplastic responses in the airway epithelial cells to inhaled radon and radon progeny and perhaps other stresses associated with ROS.

Disruption of anthrax toxin binding with the use of human antibodies and competitive inhibitors.

The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.

A nondenaturing purification scheme for the DNA-binding domain of poly(ADP-ribose) polymerase, a structure-specific DNA-binding protein.

Poly(ADP-ribose) polymerase (PARP) is thought to be involved in DNA repair given its ability to recognize and bind to DNA strand breaks. During apoptosis, PARP is proteolytically cleaved into two stable fragments, the N-terminal 25-kDa DNA-binding domain (DBD) and the 85-kDa fragment containing the automodification and catalytic domains. To understand the DNA-binding properties of PARP, we expressed a recombinant hexahistidine tagged protein (His-DBD) in Escherichia coli. We modified expression to facilitate protein folding by including zinc and reducing the induction temperature. Properly folded, the DNA-binding domain of PARP binds to single- and double-stranded DNA in a structure-specific manner. To eliminate contamination with bacterial DNA that occurred during the purification process, a purification procedure was developed to produce DNA-free protein. In addition, to remove the hexahistidine tag from the recombinant protein, thrombin cleavage was carried out while the recombinant protein was bound to a DNA column. This procedure stabilized the recombinant protein and resulted in nearly 100% cleavage with no appreciable loss to unwanted proteolytic degradation. This nondenaturing purification scheme results in high-quality, native PARP-DBD for use in structural and biochemical studies.

High-performance liquid chromatographic analysis of staurosporine in vivo. Its translocation and pharmacokinetics in rats.

The protein kinase inhibitor staurosporine (Stsp) has been used extensively to study physiological functions, biochemical mechanisms, and cancer therapy. Using an HPLC assay for Stsp developed in our laboratory, we find that only 0.7% of Stsp remains in circulating blood of rats 5 min after injection. In vitro, Stsp is adsorbed to red blood cells (RBC) weakly and reversibly. In vivo, all but 1.2-2.5% of Stsp injected is adsorbed by the heart and lungs in one passage through them, indicating that the endothelium acts as a major Stsp sink. Following initial adsorption, pharmacokinetic studies demonstrated that Stsp had a half-life of 51.6 min in plasma and 75.3 min in RBC. Thus, plasma Stsp was in the cancer therapy range of 1-10 ng/ml for 2.7 h following a bolus injection. This data indicates that a bolus injection of Stsp must be followed by a continuous infusion of low Stsp concentration for several days to produce the G1 arrest in cells necessary to stop cell proliferation.

Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase.

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.

Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints.

We investigated the requirements for protein p53 and the ATM gene product in radiation-induced inhibition of DNA synthesis and regulation of the cyclin E/ and cyclin A/cyclin dependent kinases (Cdks). Wild type (WT) mouse lung fibroblasts (MLFs), p53(-/-) knock-out MLFs, normal human skin fibroblasts (HSF-55), and human AT skin fibroblasts (GM02052) were used in the investigations. The absence of p53 had no significant effect on the inhibition or recovery of DNA synthesis throughout the S phase, as determined from BrdU labeling and flow cytometry, or the rapid inhibition of cyclin A/Cdks. Gamma radiation (8 Gy) inhibited DNA synthesis and progression into G2 during the first 3 h after irradiation, and the recovery of these processes occurred at similar rates in both WT and p53(-/-) MLFs. The cyclin A/Cdks were inhibited 55-70% at 1 h after irradiation in both cell types, but p21WAF1/Cip1 levels or p21 interaction with Cdk2 did not increase in the irradiated p53(-/-) MLFs. Although p53(-/-) MLFs do not exhibit prolonged arrest at a G1 checkpoint, radiation did induce a rapid 20% reduction and small super-recovery of cyclin E/Cdk2 within 1-2 h after irradiation. Similar inhibition and recovery of cyclin E/Cdk2 previously had been associated with regulation of transient G1 delay and the inhibition of initiation at an apparent G1/S checkpoint in Chinese hamster cells. In contrast, loss of the ATM gene product abrogated transient cyclin E/Cdk2 inhibition, most inhibition of DNA synthesis and all, but a 10-15% inhibition, of the cyclin A/Cdks. The results indicate that neither p53 nor p21 is required for transient inhibition of cyclin E/Cdk2 associated with the G1/S checkpoint or for inhibition of DNA synthesis at 'checkpoints' within the S phase. Conversely, the ATM gene product appears to be essential for regulation of the G1/S checkpoint and for inhibition of DNA replication associated with the inhibition of cyclin A/Cdk2. Differential aspects of DNA synthesis inhibition among cell types are presented and discussed in the context of S phase checkpoints.

A new mechanism for DNA alterations induced by alpha particles such as those emitted by radon and radon progeny.

The mechanism(s) by which alpha (alpha) particles like those emitted from inhaled radon and radon progeny cause their carcinogenic effects in the lung remains unclear. Although direct nuclear traversals by alpha-particles may be involved in mediating these outcomes, increasing evidence indicates that a particles can cause alterations in DNA in the absence of direct hits to cell nuclei. Using the occurrence of excessive sister chromatid exchanges (SCE) as an index of DNA damage in human lung fibroblasts, we investigated the hypothesis that alpha-particles may induce DNA damage through the generation of extracellular factors. We have found that a relatively low dose of alpha-particles can result in the generation of extracellular factors, which, upon transfer to unexposed normal human cells, can cause excessive SCE to an extent equivalent to that observed when the cells are directly irradiated with the same irradiation dose. A short-lived, SCE-inducing factor(s) is generated in alpha-irradiated culture medium containing serum in the absence of cells. A more persistent SCE-inducing factor(s), which can survive freeze-thaw and is heat labile is produced by fibroblasts after exposure to the alpha-particles. These results indicate that the initiating target for alpha-particle-induced genetic changes can be larger than a cell's nucleus or even a whole cell. How transmissible factors like those observed here in vitro may extend to the in vivo condition in the context of a-particle-induced carcinogenesis in the respiratory tract remains to be determined.

Alpha particles initiate biological production of superoxide anions and hydrogen peroxide in human cells.

The mechanism(s) by which high-linear energy transfer a particles, like those emitted by inhaled radon and radon daughters, cause lung cancer has not been elucidated. Conceivably, DNA damage that is induced by a particles may be mediated by the metabolic generation of reactive oxygen species (ROS), in addition to direct a particle-DNA interactions and hydroxyl radical-DNA interactions. Using normal human lung fibroblasts, we investigated the hypothesis that densely ionizing alpha particles may induce the intracellular generation of superoxide (O2.-) and hydrogen peroxide (H2O2). Ethidium bromide and 2',7'-dichlorofluorescein, fluorescent products of the membrane-permeable dyes hydroethidine and 2',7'-dichlorofluorescin diacetate, respectively, were used to monitor the intracellular production of O2.- and H2O2, respectively, by flow cytometry. Compared to sham-irradiated cells, fibroblasts that were exposed to alpha particles (0.4-19 cGy) had significant increases in intracellular O2.- production, along with concomitant increases in H2O2 production. Further analyses suggest that the plasma membrane-bound NADPH-oxidase is primarily responsible for this increased intracellular generation of ROS and that the ROS response does not require direct nuclear or cellular "hits" by the a particles. In this latter regard, we additionally report that unirradiated cells also show the ROS response when they are incubated with serum-containing culture medium that has been exposed to a particles or when they are incubated with supernatants from a-irradiated cells. Our overall results support the possibility that a particles, at least in part, may mediate their DNA-damaging effects indirectly via a ROS-related mechanism.

Cell cycle response to DNA damage differs in bronchial epithelial cells and lung fibroblasts.

Epithelial cells along the conducting airways can be more or less continuously exposed to DNA-damaging agents, which should limit their proliferation by inducing cell cycle checkpoints. Yet, paradoxically, airway epithelial cells frequently show a hyperplastic response when exposed to such agents. In this in vitro study, we assessed the hypothesis that normal human bronchial epithelial cells (BECs) are more resistant to the cell cycle-arresting effects of DNA damage than are human lung fibroblasts (HLFs), a cell type often investigated in the context of cell cycle checkpoints. Using ionizing radiation as a DNA-damaging insult, we have found that BECs indeed show less pronounced G1 and G2 delays than do fibroblasts. Unlike the HLFs, which ultimately enter a condition of apparently terminal arrest in the G1 phase of the cell cycle, BECs continue proliferating following their initial, transient G1 and G2 delays. Radiation-induced p53 and p21Cip1 increases were greater in HLFs than in BECs, whereas preexposure, basal levels of p53 were higher in BECs than in HLFs. The results of this investigation indicate that BECs may be less susceptible to the cell cycle-arresting effects of DNA-damaging agents, perhaps because of their higher basal levels of p53. Extension of these findings to the in vivo condition provides a possible explanation for airway epithelial cell hyperplastic responses that occur in a background of DNA-damaging stresses. Moreover, the attenuated DNA damage-induced, cell cycle checkpoint responses in BECs potentially may favor the transmission of DNA lesions to cell progeny.

The mammalian metabolite, 2-methoxyestradiol, affects P53 levels and apoptosis induction in transformed cells but not in normal cells.

The endogenous metabolite, 2-methoxyestradiol (2ME), is an inhibitor of tubulin polymerization and is therefore toxic to dividing fast-growing tumor cells. Transformed cells are not equally susceptible to the effects of 2ME. In this study the effects of 1-2 microM doses of 2ME on cell cycle progression, apoptosis induction and on p53 levels were evaluated using flow cytometry in cells with different p53 status. No effect of 2ME was seen in normal human skin fibroblast strain HSF43 with wild-type (wt) p53. However, in SV40 T antigen transformed HSF43 cells (line E8T4), 2ME caused a prominent G2/M arrest, with subsequent micronuclei formation followed by apoptosis. Increased p53 levels were present in the G2/M cells. Our results suggest that 2ME, being a microtubule poison, may release the bound p53 from T antigen, and that this p53 may enhance the apoptotic effects. Two lymphoblast cell lines derived from the same donor, TK6, expressing low levels of wt p53, and WTK1, expressing high levels of mutant p53, showed similar moderate responses to 2ME at 37 degrees C. The effects included enhanced apoptosis and a modest G2/M block. No increase in p53 levels was seen. However, at the permissive temperature of 30 degrees C marked increases in apoptosis and a prominent G2/M-phase block, similar to that seen in the E8T4 cells, were present in the WTK1 cells, indicating that the high levels of mutant p53 have now become functional, enhancing the apoptotic effects initiated by 2ME.