Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors.

Apoptosis is dependent on the activation of a group of proteolytic enzymes called caspases. Caspase activation can be detected by immunoblotting using caspase-specific antibodies or by caspase activity measurement employing pro-fluorescent substrates that become fluorescent upon cleavage by the caspase. Most of these methods require the preparation of cell extracts and, therefore, are not suitable for the detection of active caspases within the living cell. Using FAM-VAD-FMK, we have developed a simple and sensitive assay for the detection of caspase activity in living cells. FAM-VAD-FMK is a carboxyfluorescein (FAM) derivative of benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone (zVAD-FMK), which is a potent broad-spectrum inhibitor of caspases. FAM-VAD-FMK enters the cell and irreversibly binds to activated caspases. Cells containing bound FAM-VAD-FMK can be analyzed by flow cytometry, fluorescence microscopy, or a fluorescence plate reader. Using FAM-VAD-FMK, we have measured caspase activation in live non-adherent and adherent cells. We show that FAM-VAD-FMK labeled Jurkat and HeLa cells that had undergone apoptosis following treatment with camptothecin or staurosporine. Non-stimulated negative control cells were not stained. Pretreatment with the general caspase inhibitor zVAD-FMK blocked caspase-specific staining in induced Jurkat and HeLa cells. Pretreatment of staurosporine-induced Jurkat cells with FAM-VAD-FMK inhibited affinity labeling of caspase-3, -6, and -7, blocked caspase-specific cell staining, and led to the inhibition of apoptosis. In contrast, the fluorescent control inhibitor FAM-FA-FMK had no effect. Measurement of caspase activation in 96-well plates showed a 3- to 5-fold increase in FAM-fluorescence in staurosporine-treated cells compared to control cells. In summary, we show that FAM-VAD-FMK is a versatile and specific tool for detecting activated caspases in living cells.

BCL-2 is involved in preventing oxidant-induced cell death and in decreasing oxygen radical production.

It has been hypothesized that programmed cell death is mediated, in part, through the formation of free radicals via oxidative pathways. Furthermore, it has been proposed that BCL-2 acts to inhibit cell death by interfering with the production of oxygen-derived free radicals induced by a wide variety of stimuli. In order to examine the antioxidant function of BCL-2, we transfected mouse epidermal cells JB6 clone 41 with the expression vector pD5-Neo-BCL-2 and studied the effect of BCL-2 overexpression on oxidant-induced cell death and on the production of reactive oxygen species. Compared to Neo control cells, BCL-2-expressing cells are more resistant to the killing and growth retardation induced by hydrogen peroxide, superoxide, or by the oxygen radical-generating quinone-containing compounds menadione, diaziquone and adriamycin. The latter compounds generate reactive oxygen species during bioreductive metabolism. In addition, the exposed cells die by necrosis rather than apoptosis. Hydroxyl radical levels generated by the quinone-containing agents were low in BCL-2-expressing JB6 cells compared to control Neo cells. BCL-2, however, does not change the activities of the major cellular antioxidant enzymes superoxide dismutase, catalase or glutathione peroxidase. On the other hand, the glutathione concentrations increased in BCL-2 overexpressing cells after oxidative challenge, while the opposite was true for control cells. Thus, our results suggest that BCL-2 inhibition of oxidant-induced cell death is mediated, at least in part, through an antioxidant pathway, and that this pathway involves glutathione.

Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: oxyradical overload diseases.

Hemochromatosis and Wilson disease (WD), characterized by the excess hepatic deposition of iron and copper, respectively, produce oxidative stress and increase the risk of liver cancer. Because the frequency of p53 mutated alleles in nontumorous human tissue may be a biomarker of oxyradical damage and identify individuals at increased cancer risk, we have determined the frequency of p53 mutated alleles in nontumorous liver tissue from WD and hemochromatosis patients. When compared with the liver samples from normal controls, higher frequencies of G:C to T:A transversions at codon 249 (P < 0.001) and C:G to A:T transversions and C:G to T:A transitions at codon 250 (P < 0.001 and P < 0.005) were found in liver tissue from WD cases, and a higher frequency of G:C to T:A transversions at codon 249 (P < 0.05) also was found in liver tissue from hemochromatosis cases. Sixty percent of the WD and 28% of hemochromatosis cases also showed a higher expression of inducible nitric oxide synthase in the liver, which suggests nitric oxide as a source of increased oxidative stress. A high level of etheno-DNA adducts, formed from oxyradical-induced lipid peroxidation, in liver from WD and hemochromatosis patients has been reported previously. Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to 4-hydroxynonenal, an unsaturated aldehyde involved in lipid peroxidation, and observed an increase in G to T transversions at p53 codon 249 (AGG to AGT). These results are consistent with the hypothesis that the generation of oxygen/nitrogen species and unsaturated aldehydes from iron and copper overload in hemochromatosis and WD causes mutations in the p53 tumor suppressor gene.

Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca2+ mobilization.

The mechanism by which Bcl-2 inhibits cell death is unknown. It has been suggested that Bcl-2 functions as an antioxidant. Because Bcl-2 is localized mainly to the membranes of the endoplasmic reticulum (ER) and the mitochondria, which represent the main intracellular storage sites for Ca2+, we hypothesized that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca2+ homeostasis. To test this hypothesis, we examined the effect of oxidant treatment on viability in normal rat kidney (NRK) cells and in NRK cells stably transfected with Bcl-2 in the presence or absence of intracellular Ca2+, and we compared the effect of Bcl-2 expression on oxidant-induced intracellular Ca2+ mobilization and on ER and mitochondrial Ca2+ pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant to H2O2-induced cytotoxicity than control cells. EGTA-AM, an intracellular Ca2+ chelator, as well as the absence of Ca2+ in the medium, reduced H2O2-induced cytotoxicity in both cell lines. Compared with controls, cells overexpressing Bcl-2 showed a delayed rise in intracellular Ca2+ concentration ([Ca2+]i) after H2O2 treatment. After treatment with the Ca2+ ionophore ionomycin, Bcl-2-transfected cells showed a much quicker decrease after the maximal rise than control cells, suggesting stronger intracellular Ca2+ buffering, whereas treatment with thapsigargin, an inhibitor of the ER Ca2+-ATPases, transiently increased [Ca2+]i in control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation show that NRK-Bcl-2 cells have a higher capacity for mitochondrial Ca2+ storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity of mitochondria to store Ca2+.

bcl-2 enhancement of malignant transformation in mouse epidermal JB6 cells.

Increased bcl-2 expression is a common feature of many types of human malignancies, which implies that bcl-2 plays an important role in tumorigenesis. To better understand the molecular mechanisms of bcl-2-induced oncogenesis, we examined the effects of bcl-2 expression on transformation of mouse epidermal JB6 cells induced by the tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA). Promotion-sensitive JB6 clone41 cells were transfected with the bcl-2-containing expression vector pD5-neo/bcl-2, and the soft agar growth of bcl-2-transfected cells and control cells were compared. bcl-2 overexpression in JB6 clone41 cells caused a TPA-induced soft-agar growth fivefold greater than the growth of nontransfected or vector-transfected (neo control) cells. bcl-2 expression in the absence of TPA did not lead to colony formation in soft agar. Because the level of the transcription factor activator protein 1 (AP-1) has been shown to be critical for the responsiveness of JB6 cells to TPA-induced transformation, we compared c-jun and c-fos expression as well as the AP-1-binding activity and the AP-1-mediated transactivation of the reporter construct TRE-CAT between bcl-2-expressing cells and control cells. When compared with control cells, bcl-2-transfected cells expressed significantly more c-fos but not c-jun after TPA treatment. Furthermore, the levels of AP-1 and AP-1-induced transactivation of TRE-CAT were greater in bcl-2-transfected cells than in control cells after TPA treatment. These results showed that bcl-2 cooperates with a tumor promoter such as TPA in the induction of malignant transformation in mouse epidermal cells and that bcl-2 enhances soft-agar growth by stimulating signaling pathways that led to increased AP-1 expression.

Manganese superoxide dismutase expression inhibits soft agar growth in JB6 clone41 mouse epidermal cells.

Manganese superoxide dismutase (MnSOD) has been found to be depleted in a variety of tumor cells as well as in in vitro transformed cell lines, suggesting that MnSOD may function as an anticarcinogen by protecting the cell from oxidant-induced carcinogenesis. The relationship between MnSOD expression and tumor promotion was studied by transfection of a human MnSOD cDNA into the promotable mouse epidermal cell line JB6 clone41. The effect of MnSOD overexpression on the promotion-sensitive phenotype of JB6 cells was assessed by measuring growth characteristics such as growth rate and the ability to form colonies in soft agar. Compared with the parental and vector-transfected (gpt) control cells, MnSOD-overexpressing cells had a slower growth rate and their ability to form colonies in soft agar was significantly decreased in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Since the transformation-sensitive phenotype of JB6 clone41 cells is associated with increased expression of the transcription factor AP-1, we compared c-jun and c-fos mRNA expression in MnSOD-transfected and control JB6 cells. Overexpression of MnSOD led to a significant decrease in c-jun and c-fos expression in response to treatment with TPA or the oxidant promoter superoxide. These findings indicate that the promotion-sensitive phenotype of JB6 clone41 cells can be reverted by increasing MnSOD intracellularly. A possible mechanism is that elevated MnSOD expression might change the intracellular redox state by altering the balance of reactive oxygen species. This could lead to a modulation of TPA and oxidant-induced signal transduction pathways controlling cell growth and differentiation.

Ultraviolet-B-light-induced mutagenesis of C-H-ras codons 11 and 12 in human skin fibroblasts.

Mutations in the ras oncogene are detected with a high frequency in non-melanoma skin cancer. Approximately half of the squamous-cell carcinomas (SCC) and one third of the basal-cell carcinomas (BCC) carry mutations at the second position of Ha-ras codon 12 (GGC to GTC), whereas mutations in Ki-ras codon 12 occur less frequently. Since the mutations in the Ha-ras and Ki-ras oncogenes are located opposite potential pyrimidine dimer sites (C-C), it is likely that the mutations are induced by ultraviolet radiation present in sunlight. We studied the capacity of ultraviolet B (UVB) light to induce base-pair changes in Ha-ras codons 11 and 12 in human skin fibroblasts. UVB induced mostly C to T and G to A transitions and C to A and G to T transversions. The base-pair change with the highest relative abundance was C to T in the middle position of codon 11 followed by (in diminishing relative abundance) C to A in the middle position of codon 11, G to A and G to T in the middle position of codon 12. The C to T and G to A transitions are compatible with pyrimidine photodimers as pre-mutagenic lesions, whereas the C to A and G to T transversions could be generated due to the formation of 8-hydroxyguanine, which is the major oxidation product of guanine. The relative abundance of mutations induced by UVB in Ha-ras codons 11 and 12 does not correlate with mutations observed in the DNA from non-melanoma skin cancer, where the G to T transversion in the middle position of codon 12 is selected.

Benzo[a]pyrene-induced mutagenesis of p53 hot-spot codons 248 and 249 in human hepatocytes.

Human tobacco-related cancers show a high frequency of G-to-T transversions in several mutation hot-spot regions of the p53 tumor suppressor gene, probably the result of specific mutagens in tobacco smoke, most notably benzo[a]pyrene. To gain insight into the mechanism of formation of these G-to-T transversions in tobacco-associated carcinogenesis, we studied the mutagenesis of p53 codons 247-250 by benzo[a]pyrene in human hepatocellular carcinoma cells by restriction fragment length polymorphism-polymerase chain reaction genotypic analysis. Benzo[a]pyrene preferentially induced G-to-T transversion in the second and third positions of codon 248 and C-to-A transversion in the first position of codon 248. However, benzo[a]pyrene did not induce base-pair changes in codon 249, which is a mutational hot-spot in aflatoxin-related hepatocarcinogenesis, in which predominantly G-to-T transversion in the third position of codon 249 is observed. The benzo[a]pyrene-induced G-to-T transversion in the middle position of codon 248, in which arginine is changed into leucine, is frequently observed in tumors of the lung. The other two benzo[a]pyrene-induced base-pair changes in codon 248, namely the C-to-A transversion in the first position and G-to-T transversion in the third position, do not lead to a change in the amino-acid composition of the p53 protein. These mutations are silent and therefore are not selected in tumors. It follows that benzo[a]pyrene-induced mutability on the DNA level in p53 codons 247-250 correlates well with the type of mutation found in tumors of the lung. Therefore, our results support the hypothesis that benzo[a]pyrene is the etiological agent in tobacco-related cancers.

Mechanism of induction of c-fos by ultraviolet B (290-320 nm) in mouse JB6 epidermal cells.

The UVB (290-320 nm) portion of the solar spectrum possesses the highest activity for the induction of skin cancer and has the capacity to stimulate epidermal proliferation. We report that UVB is a transcriptional inducer of the c-fos protooncogene in mouse JB6 epidermal cells. Induction is biphasic with an immediate early peak at 30-60 min and a second broader peak 8 h following irradiation. The immediate early phase is suppressed by inhibitors of nuclear adenosine diphosphoribose transferase. For UVB induction, the formation of full-length messages is less efficient than of early, short messages, while both types of messages are produced at similar rates following serum stimulation. Experiments with stable transfectants with reporter constructs linked to 5'-upstream sequences of c-fos indicate that UVB and serum stimulation both require the sequences from -345 to -285 which contain the joint DSE-AP-1 enhancer motifs for efficient induction. Mobility shift data reveal that the complement of c-Fos and c-Jun proteins which bind to the fos-AP-1 octanucleotide decrease immediately following irradiation. Increased binding of Fos and Jun is observed 8-24 h later. UVB did not cause an observable change in the nuclear proteins which bind to the dyad symmetry element oligonucleotide in vitro. Fos protein was detected among the binding proteins. We propose that the two phases of UVB-induced c-fos expression occur by quite different mechanisms. The immediate early phase is inhibited by adenosine diphosphoribose transferase inhibitors because poly-ADP ribosylation of chromosomal proteins is required for the resealing of UVB-induced DNA strand breaks which otherwise retard message elongation. The production of an autocrine factor may be responsible for the late phase of c-fos induction.

Mechanism of c-fos induction by active oxygen.

We have compared the mechanisms of the transcriptional induction of c-fos in mouse epidermal cells JB6 (clone 30) by an extracellular burst of active oxygen of the type produced by inflammatory phagocytes to induction by serum and phorbol ester. All three inducers elicit a characteristic immediate early response of c-fos which is inhibited by the protein kinase inhibitor H7 but enhanced by the protein synthesis inhibitor cycloheximide. Experiments with stable transfectants containing fos 5' upstream regulatory sequences linked to an HSV-tk-chloram-phenicol-acetyl-transferase reporter construct indicate that the joint dyad symmetry element-AP-1 motifs exert the most potent enhancer effect in response to active oxygen as well as serum. It is concluded that the different signal transduction pathways used by these inducers converge to the same 5' regulatory sequences of c-fos. In contrast to these common features only active oxygen induction of c-fos required the poly-ADP-ribosylation of chromosomal proteins. The inhibitors of ADP-ribose transferase benzamide and 3-amino-benzamide suppressed the elongation of the c-fos message and the de novo synthesis of nuclear factors, among them c-Fos and c-Jun, which bind to the fos-AP-1 motif in vitro only following stimulation with active oxygen. No active oxygen-induced change was observed in the protein complex which binds to an oligonucleotide containing the SIF and dyad symmetry element motifs in vitro. The presence of Fos and Jun proteins was detected in this complex. Only active oxygen, but not serum or phorbol ester, induces DNA breakage. We propose that poly-ADP-ribosylation is required because it participates in the repair of DNA breaks which interfere with transcription. We observed that Fos protein is weakly poly-ADP-ribosylated in response to active oxygen, but the functional role of this modification remains unclear.

Constitutive and phorbol-myristate-acetate regulated antioxidant defense of mouse epidermal JB6 cells.

Because oxidative processes can participate in tumor promotion, it is likely that the cellular antioxidant defense also plays a role. We have compared the levels of the three major antioxidant enzymes, Cu,Zn-superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), in promotable mouse epidermal JB6 cells clone 41 and nonpromotable cells, clone 30. We found that the constitutive activities of SOD and catalase were approximately twice as high in clone 41 as in clone 30 while the GPx activities were comparable. Correspondingly, catalase protein concentrations were higher in clone 41, according to immunoblots. Northern blot analysis indicated that the steady-state mRNA concentrations for SOD and catalase, but not for GPx, were considerably higher in clone 41 than in clone 30. Southern blot analysis showed no difference between the two clones in their complements of the SOD and catalase genes. Clone 41 also contained slightly higher constitutive levels of glutathione. The higher antioxidant capacity of promotable clone 41 may protect it from excessive toxicity of oxidant promoters and allow growth stimulation. Certain tumor promoters that lack oxidizing properties may generate a cellular prooxidant state by a variety of mechanisms (e.g., it had been reported that the phorbol ester PMA decreases the activities of catalase and SOD in mouse skin). We found for JB6 cells that this loss of enzyme activity was due to a decrease in the steady-state concentrations of catalase and SOD mRNA. No significant changes in the rates of transcription were detected in nuclear run-off experiments. The observed decreases in catalase and SOD can be considered as part of the complex reprogramming of gene expression that is set in motion by phorbol-12-myristate-13-acetate.

Removal of aflatoxin B1-DNA adducts and in vitro transformation in mouse embryo fibroblasts C3H/10T1 1/2.

The mechanism of in vitro transformation of the mouse embryo fibroblast C3H/10T 1/2 clone 8 by aflatoxin B1 (AFB1) was studied in confluent holding (CH) experiments. Confluent cultures of C3H/10T 1/2 cells were treated with AFB1 for 16 hours, and the DNA adduct composition and concentration were determined by chromatographic procedures after 0, 8, 16, and 40 hours of CH when the cells were replated at low density for the expression of their colony-forming ability and the formation of transformed foci. Total adduct concentration and the concentration of the major primary adduct 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua) decreased continuously during CH due to spontaneous decomposition and probably also due to enzymatic repair processes. In contrast, the more chemically stable secondary product 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-triamino-Py) accumulated in the DNA and reached its maximum concentration after 16 hours of CH. While the loss of total AFB1-DNA adducts during CH was reflected in recovery of viability, the potential to form transformed foci reached a maximum after 16 hours of CH and then decreased with continued CH below the initial value. Therefore, no simple relationship exists between the concentration of the total adducts AFB1-N7-Gua and AFB1-triamino-Py at the time of release from CH and the potential to form transformed foci. However, DNA lesions or abnormal DNA configurations formed during CH as a consequence of the cellular processing of AFB1-DNA adducts may play a role in the transformation process.