Photometric Enzyme Immunoassay Research Articles

Competitive photometric enzyme immunoassay for fullerene C60 and its derivatives using a fullerene conjugated to horseradish peroxidase

The article describes a highly sensitive single-step microplate enzyme immunoassay of the ELISA type for fullerene C60 and its derivatives. Monoclonal anti-fullerene antibodies and a conjugate between fullerene and horseradish peroxidase were used as specific reagents. A direct competitive ELISA was carried out that was based on antibodies immobilized in the well of a microtiter plate, a peroxidase-labeled antigen, and detection via the dye formed from 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. Both pristine fullerene C60 and its water-soluble forms can be determined. The detection limits are 1.5 ng∙mL−1 for fullerene C60, and between 0.1 and 1.3 ng∙mL−1 for its derivatives. This ELISA format allows for almost two-fold reduction of the time needed for the assay in comparison to indirect scheme with labeled antibodies.

Feed contaminated with Fusarium toxins alter lymphocyte proliferation and apoptosis in primiparous sows during the perinatal period

Two groups of pregnant primiparous sows (day 89±2 of gestation) were 54days (±1day) fed either with an experimental diet (5.08mgkg−1 deoxynivalenol – DON, 0.09mgkg−1 zearalenone and 21.61mgkg−1 fusaric acid) or control diet (0.25mgkg−1 DON). The consummation of feed was significantly higher in the control group. Lymphocyte stimulation in culture from peripheral blood lymphocytes (PBL) was measured by BrdU incorporation test using two different concentrations of mitogen PHA 10 and 20μgml−1, two different concentrations of DON (5 and 1μgml−1) and a combination of both, PHA and DON (PHA 10+DON 5, PHA 10+DON 1 and PHA 10+DON 0.1μgml−1). The lymphocyte proliferation, except for PHA 10μgml−1, was significantly higher in the experimental group. Further on, using the photometric enzyme immunoassay, the apoptosis was studied in non-stimulated 72h lymphocyte culture or stimulated with 1μgml−1 of DON. The significantly higher apoptosis was in non-stimulated lymphocyte cultures from the experimental group (P=0.036). The results suggest that such feed may affect the peripheral lymphocyte population in the direction of their proliferation response and programmed cell death.

Can Gene Methylation, Telomere Length and Activity of Telomerase Help with Individual Risk and Treatment Strategy of MDS Patients?

Background: Epigenetic de novo methylation of CpG-rich areas located in the promotors of gene p15INK4b (CDKN2B) as it is capable to silence tumor suppressor genes and knowledge of telomere-telomerase complex may bring an important sign into molecular backround of malignant transformation in patients with myelodysplastic syndromes (MDS).Aims: Intensity of gene methylation, the variability of telomere length (TRF) and telomerase activity (TA) were studied in bone marrow (BM) or peripheral blood (PB) cells of patients with MDS before treatment and during the course of the disease to evaluate its prognostic significance and association with clinical outcomes and disease progression.Methods: 110 patients with MDS were stratiffied according to the WHO classification: 18 × RA, 3 × RARS, 32 × RCMD, 12 × 5q-syndrome, 17 × RAEB1, 19 × RAEB2 and 9 × MDS/MPS. BM cells from 30 age matched healthy donors served as controls, 5 leukemic cell lines were examined as positive controls. The level of DNA methylation was quantified by methylation specific PCR after sodium bisulphate modification of DNA, CpGenom DNA Modification kit from Scintila. The methylation status was dedicated as Methylation Indices (MI) in the range 0.0–1.0. The telomere length was determined as terminal restriction fragment (TRF index in kbp) after Southern analysis of genomic DNA, TeloTAGGG Telomere Length Assay kit from Roche. The activity of telomerase was assessed by Quantitative TRAP assay based on a photometric enzyme immunoassay on solid phase, Telo TAGGG Telomerase PCR Elisa plus kit from Roche. We postulated TRF shorter than 7.5 kbp as reduced telomeres and values of TA higher than 0.0263 as positive telomerase activity.Results: Aberrant methylation of CDKN2B gene was present in 64% of MDS untreated patients showing significant heterogeneity in WHO subgroups. Mean values of MI increased as follows: from 0.18 ± 0.13 in RCMD, 0.21 ± 0.11 in RA/RARS, 0.26 ± 0.19 in 5q- syndrome, 0.33 ± 0.17 in RAEB1, 0.34 ± 0.17 in RAEB2, up to 0.41 ± 0.14 in MDS/MPS. A significant difference was found between early forms of MDS (RA/RARS, RCMD, 5q-syndrome) and advanced MDS with excess of blasts (RAEB1, RAEB2, MDS/MPS). High level of methylation CDKN2B gene was observed in 70% (33/47) of patients with advances forms of MDS (MI=0.36 ± 0.17) in comparison with early phases of MDS (MI=0.20 ± 0.14) in 60% (39/65) of patients. In one patient with RAEB2, a significant decrease in MI from 0.44 in BM and 0.37 in PB to 0,25 to 0,04, respectively was observed after 3 months of treatment with decitabine together with simultaneous prolongation of TRF from 7,62 kbp to 10.5 kbp. The patient achieved partial remission of the disease after 6 months of treatment with corresponding values of MI=0.20 and TRF=9.63kbp. In another patient, a progression of the disease from RCMD towards RAEB was connected vith increase in MI from 0.00 to 0.39 that correlated with elevation of telomerase activity from 0 to 0.0534.Conclusions: The results confirm association of aberrant gene methylation with progression of early MDS to advanced forms with excess of blasts. Our findings also showed a certain degree of correlation between molecular changes in gene methylation, telomere length and telomerase activity in different phases of disease. Thus, determination of these markers may contribute to more precise estimation of an individual MDS patient's risk and for decision of an optimal treatment strategy.

The effect of progesterone, testosterone and synthetic progestogens on growth factor- and estradiol-treated human cancerous and benign breast cells

Objective The effects of progesterone (P), testosterone (T), chlormadinone acetate (CMA), medroxyprogesterone acetate (MPA), norethisterone (NET), levonorgestrel (LNG), dienogest (DNG), gestodene (GSD) and 3-ketodesogestrel (KDG) were investigated in normal human breast epithelial MCF10A cells. In addition, the effects of these steroids were tested in estrogen and progesterone receptor positive HCC1500 human primary breast cancer cells. Study design MCF10A cells were incubated with each progestogen at 1 μM and 100 nM for 7 days with growth factors epidermal growth factor (EGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF-I) (GFs, each 1 pM), and HCC1500 cells with GFs and/or estradiol (E2) 100 pM. Cell proliferation rate was measured by ATP-assay and cell death by photometric enzyme immunoassay. Ratios of cell death:proliferation were calculated. Results MPA and CMA with GFs induced proliferation of MCF10A cells. P, T, NET, LNG, DNG, GSD and KDG had no significant effect. In HCC1500 cells, MPA and CMA with GFs had an inhibitory effect compared to GFs alone. NET, LNG, DNG, GSD, KDG and T enhanced the proliferative effect of GFs. P had no significant effect. No progestogen could further enhance the stimulatory effect of E2 on HCC1500 cells; all but KDG inhibited it. MPA, GSD, T, CMA and NET had an anti-proliferative effect on the mitotic GF and E2 combination. P, LNG, DNG and KDG had no significant effect. Conclusions Estrogens and mitogenic growth factors from stromal breast tissue are significant in growth-regulation of breast cells and may alter responses to progestogens. Certain progestogens are able to induce proliferation of or inhibit growth of benign or malignant human breast epithelial cells independently of the effects of growth factors and E2; therefore, choice of progestogen for hormone therapy may be important in terms of influencing possible breast cancer risk.

Apoptosis in Thalassemia major Reduced by a Butyrate Derivative

Background and Objectives: In cases of β-thalassemia major, apoptosis appears to be greatly enhanced in the early-stage erythroid precursors in the bone marrow leading to ineffective erythropoiesis. L-Carnitine is found to strongly reduce apoptosis in different diseases. We investigated the effect of oral L-carnitine therapy on apoptosis in thalassemia major patients. Methods: Eighteen thalassemia major patients with a mean age of 12.2 ± 6.6 years were included. Detection of apoptosis was done by photometric enzyme immunoassay (ELISA) and agarose gel electrophoresis before and after 6 months of oral therapy with L-carnitine (50 mg/kg/day). Results: A significant decrease of apoptosis frequency in the erythroid precursors in the bone marrow of studied cases was noted after therapy. The quantity of nucleosomes measured by ELISA dropped from 3.65 ± 1.338 to 1.60 ± 0.65 after therapy (p = 0.005). A positive ladder pattern reflecting apoptosis on agarose gel electrophoresis was detected in 88.9% of cases prior to treatment versus 16.7% after therapy (p = 0.006). Patients also had a significant decrease in the frequency of transfusions and increase in the pre-transfusion hemoglobin levels after therapy. Conclusion:L-Carnitine seems to be a good modulator of apoptotic processes in thalassemic patients leading to a decreased frequency of programmed erythroblast death and general improvement of the disease condition.

The effects of progesterone, medroxyprogesterone acetate, and norethisterone on growth factor- and estradiol-treated human cancerous and noncancerous breast cells

Mitogenic growth factors from stromal breast tissue and estrogens are important in growth regulation of breast cells and may modify different responses dependent on the choice of progestogens. The effects of the acknowledged pharmacologically significantly different progestogens, progesterone, medroxyprogesterone acetate (MPA), and norethisterone, were investigated in breast cells treated with growth factor and/or estradiol, comparing normal and primary cancer cells for the first time. MCF10A (human epithelial, estrogen-receptor-negative and progesterone-receptor-negative normal breast cells) were incubated with progestogens at 1 microM and 100 nM for 7 days and epidermal growth factor, fibroblast growth factor, and insulin-like growth factor at 1 pM. HCC1500 (human estrogen-receptor-positive and progesterone-receptor-positive primary breast cancer cells) were incubated in the same way with growth factors and/or estradiol at 100 pM. Cell proliferation rate was measured by the ATP assay, whereas cell death was measured by photometric enzyme immunoassay. Ratios of cell death to proliferation were calculated from these results. In combination with growth factors, MPA reduced the ratio in favor of a proliferative effect in MCF10A cells, followed by norethisterone. Progesterone had no significant effect on the growth factor response. In HCC1500 cells, MPA with growth factors increased the ratio favoring inhibition, norethisterone had a proliferative effect, and progesterone had no significant effect. In combination with estradiol, MPA increased the ratio to inhibition to the greatest extent, followed by norethisterone and progesterone at 1 muM. In combination with growth factors and estradiol, MPA and norethisterone both showed an inhibitory effect, whereas progesterone had no significant effect. Certain progestogens are able to induce the proliferation of benign or malignant human breast cells independently of the effects of growth factors and estradiol. Therefore, the choice of progestogen may be important in terms of influencing a possible breast cancer risk.

L-Carnitine and Apoptosis in Thalassemia Major Patients.

Background and Objectives:Thalassemia major patients suffer from severe anemia due to intramedullary hemolysis of erythroid progenitors. Depending on the severity of their disease, thalassemic patients exhibit different patterns of apoptosis. In cases of β-thalassemia major, apoptosis appears to be greatly enhanced in the early-stage erythroid precursors in the bone marrow and it is considered to be the machinery via which ineffective erythropoiesis develops. L-Carnitine is a butyrate derivative found to strongly reduce apoptosis in different diseases. We investigated the effect of oral L-carnitine therapy on apoptosis in thalassemia major patients.Methods:eighteen Thalassemia major patients were included. Detection of apoptosis was done by Photometric enzyme immunoassay (Elisa) and agarose gel electrophoresis before and after 6 months oral therapy with L-Carnitine (50mg/kg/day).Results:The mean age was 12.2±6.6 years. All patients tolerated L-carnitine with no side effects. A significant decrease of apoptosis rates in the erythroid precursors in the bone marrow of studied cases was noted after therapy by. The enrichment factor measured by the photometric enzyme immunoassay dropped from 3.65±1.338 before therapy to 1.60±0.65 after therapy (p=0.005). A positive ladder pattern reflecting apoptosis on agarose gel electrophoresis was detected in 88.9% of cases prior to treatment (figure 1) versus 11.1% after therapy (figure 2) (p=0.006). Patients also had significant decrease in the frequency of transfusions and increase in the pre-transfusion hemoglobin levels after therapy. These results were found to significantly correlate with the reduction in apoptosis rates (p=0.006, r = 0.543 and p=0.027 &r = −0.7762 respectively).Conclusion:Apoptosis plays a major role in the underlying pathophysiology of anemia in thalassemic patients. L-Carnitine seems to be a good modulator of apoptotic processes in such patients leading to decreased rates of programmed erythroblast death and general improvement of the disease condition. Further large-scale studies are needed to evaluate the overall role of L-Carnitine in the management of these patients. [Display omitted]

In vivo TNF-alpha inhibition ameliorates cardiac mitochondrial dysfunction, oxidative stress, and apoptosis in experimental heart failure.

Heart failure is associated with increased myocardial expression of TNF-alpha. However, the role of TNF-alpha in the development of heart failure is not fully understood. In the present study, we investigated the contribution of TNF-alpha to myocardial mitochondrial dysfunction, oxidative stress, and apoptosis in a unique dog model of heart failure characterized by an activation of all of these pathological processes. Male mongrel dogs were randomly assigned (n = 10 each) to 1) normal controls; 2) chronic pacing (250 beats/min for 4 wk) with concomitant administration of etanercept, a soluble p75 TNF receptor fusion protein, 0.5 mg/kg subcutaneously twice weekly; 3) chronic pacing with administration of saline vehicle. Mitochondrial function was assessed by left ventricular (LV) tissue mitochondrial respiratory enzyme activities. Oxidative stress was assessed with aldehyde levels, and apoptosis was quantified by photometric enzyme immunoassay for cytoplasmic histone-associated DNA fragments and terminal deoxynucleotide transferase-mediated nick-end labeling (TUNEL) assays. LV activity levels of mitochondrial respiratory chain enzyme complex III and V were reduced in the saline-treated dogs and restored either partially (complex III) or completely (complex V) in the etanercept-treated dogs. Aldehyde levels, DNA fragments, and TUNEL-positive cells were increased in the saline-treated dogs and normalized in etanercept-treated dogs. These changes were accompanied by an attenuation of LV dilatation and partial restoration of ejection fraction. Our data demonstrate that TNF-alpha contributes to progressive LV dysfunction in pacing-induced heart failure, mediated in part by a local impairment in mitochondrial function and increase in oxidative stress and myocyte apoptosis.

Role of p53 in G2/M cell cycle arrest and apoptosis in response to γ-irradiation in ovarian carcinoma cell lines

We investigated the cell cycle and apoptotic response to irradiation in 4 human ovarian carcinoma cell lines, i.e., PA-1, Caov-3, SK-OV-3, and ES-2. Cell lines were also analysed for their p53 and Bax expression to address the relationship with cell cycle and apoptotic response. Apoptosis was examined by flow cytometric measurement of annexin V binding and by determination of cytoplasmic histone-associated DNA fragments with a photometric enzyme immunoassay. Cell cycle analyses were performed on the basis of flow cytometry. p53 and Bax protein expression was examined by immunocytochemistry in untreated cells and after irradiation. p53 cDNA sequencing and a functional yeast-based assay (FASAY) were performed to determine the p53 mutational status. All cell lines exhibited a dose-dependent G2/M arrest. No arrest in G1 was seen. A strong correlation was found between the G2/M arrest and the induction of apoptosis. PA-1, the only cell line found to express wild-type p53, showed the highest susceptibility to accumulate in G2/M and the strongest apoptotic response after irradiation. In this cell line irradiation resulted in an unequivocal accumulation of p53 protein and in an increased expression of Bax protein. Caov-3, lacking wild-type p53, showed upregulation of Bax expression after irradiation. Caov-3 proved to be relative sensitive to apoptosis compared to SK-OV-3 and ES-2. These two cell lines were found to be p53 mutated in sequence analysis and irradiation had no effect on the expression of p53. No change in Bax expression was seen in ES-2, while SK-OV-3 exhibited decreased Bax protein levels after irradiation. Our data suggest that the G2/M arrest is an important component of the pathway leading from irradiation-induced DNA damage to apoptosis in the examined cell lines. The G2/M arrest and associated apoptosis found in the examined cell lines does not necessarily require wild-type p53, although wild-type p53 and possibly Bax may contribute to a maximum response to irradiation. Two independent mechanisms, p53-dependent and p53-independent, are suggested in the examined cell lines.

Secretory phospholipase A2 and phospholipids in neural membranes in an experimental epilepsy model.

Previous studies have revealed an increase in several forms of phospholipase A2 activity associated with cell injury, but the secretory form of phospholipase A2 has not previously been studied in neurological disorders. We investigated the influence of seizures on secretory phospholipase A2 and phospholipid breakdown in synaptosome fractions prepared from rat hippocampus, cortex and cerebellum in pentylenetetrazol-induced epilepsy. Secretory phospholipase A2 concentration was measured by a photometric enzyme immunoassay. The synaptosomes underwent extraction, and the phospholipids fractions for phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylcholine were recovered from the thin layer chromatography plates. The amount of each phospholipid was quantified using the amount of recovered phosphate in each phospholipid spot. Secretory phospholipase A2 concentration was found to be significantly higher in the epileptic group when compared with the control group. The amounts of phospholipids were found to be highly variable in different brain regions. Our results suggest that epileptic seizures enhanced phospholipid breakdown and induced alterations in the distribution of phospholipids in different brain regions.

Murine Bone Marrow-Derived Mast Cells Exhibit Evidence of Both Apoptosis and Oncosis After IL-3 Deprivation

IL-3 deprivation has been reported to induce apoptosis of bone marrow-derived mast cells. In order to evaluate this type of cell death further, we employed trypan blue and propidium iodide stainings, photometric enzyme immunoassay, fluorescence measurement of caspase-3, DNA electrophoresis, flow cytometry and transmission electron microscopy. In this experiment, although several evidences supporting apoptosis were demonstrated some findings were not consistent with typical apoptosis. On the other hand, electron microscopical observation demonstrated that most cells from all the time phases after IL-3 deprivation showed the morphology of typical oncosis, i.e. cell swelling, disintegration of ultrastructure and subsequent karyolysis. Only a small number of cells from the later time phases showed apoptotic morphology. We here suggest that BMMCs undergo both apoptosis and oncosis after IL-3 deprivation and that the dominant type of prelethal change is oncosis in all time phases, although apoptosis also plays a partial role in the late time phases.

Murine Bone Marrow-Derived Mast Cells Exhibit Evidence of Both Apoptosis and Oncosis After IL-3 Deprivation

IL-3 deprivation has been reported to induce apoptosis of bone marrow-derived mast cells. In order to evaluate this type of cell death further, we employed trypan blue and propidium iodide stainings, photometric enzyme immunoassay, fluorescence measurement of caspase-3, DNA electrophoresis, flow cytometry and transmission electron microscopy. In this experiment, although several evidences supporting apoptosis were demonstrated some findings were not consistent with typical apoptosis. On the other hand, electron microscopical observation demonstrated that most cells from all the time phases after IL-3 deprivation showed the morphology of typical oncosis, i.e. cell swelling, disintegration of ultrastructure and subsequent karyolysis. Only a small number of cells from the later time phases showed apoptotic morphology. We here suggest that BMMCs undergo both apoptosis and oncosis after IL-3 deprivation and that the dominant type of prelethal change is oncosis in all time phases, although apoptosis also plays a partial role in the late time phases.

Genistein produces reduction in growth and induces apoptosis of rat RPE-J cells

Purpose. To investigate the effect of the tyrosine kinase inhibitor, genistein, on the growth of the retinal pigment epithelial (RPE) cell. Methods. The tyrosine kinase inhibitor, genistein, was administered in culture to the rat retinal pigment epithelial cell line, RPE-J. The effect on cell viability and growth was assessed by trypan blue dye exclusion. Induction of apoptosis was confirmed morphologically by light and electron microscopy and oligonucleosomal fragmentation was assessed by TUNEL and DNA ladder. Quantitation was undertaken by propidium iodide staining and photometric enzyme immunoassay. Western blot was performed to study poly-(ADP-ribose)-polymerase cleavage (PARP). To confirm the involvement of caspase, the caspase inhibitor z-VAD-fmk was employed. In addition, cell cycle phase was determined by flow cytometry. Results. We here demonstrate that genistein treatment of RPE-J cells produces a dose- and time-dependent growth inhibition. Genistein in higher concentration induces apoptosis of rat RPE-J cell. z-VAD-fmk inhibited this type of apoptosis and cleavage of PARP enzyme was demonstrated. Ten micromolar genistein inhibited cell proliferation by G 0 /G 1 arrest without inducing apoptosis of the major population. Whereas 50 µM genistein caused growth inhibition of RPE-J cells by G 2 /M arrest and subsequent apoptotic death. Conclusions. Genistein inhibits RPE cell growth and induces apoptosis. The ability of genistein to inhibit the proliferation and to induce apoptosis of RPE cells could be potentially therapeutic for proliferative vitreoretinopathy.

Enzyme-linked immunosorbent assay detection of prostate-specific antigen messenger ribonucleic acid in prostate cancer

Objectives. To develop a rapid, sensitive, reverse transcriptase-polymerase chain reaction (RT-PCR) prostate-specific antigen (PSA) messenger ribonucleic acid (mRNA) detection method by applying colorimetric enzyme-linked immunosorbent assay (ELISA). Methods. Total RNA was extracted from 16 urogenital cancer cells (including PSA-producing LNCaP cells) from pelvic and inguinal lymph node aspiration biopsy samples from patients with prostate, bladder, and penile cancer, as well as from blood samples of 500 patients with urogenital cancer. We used r Tth polymerase for RT and PCR. The RNA target was amplified by RT-PCR with dinitrophenyl-labeled primer. The PCR product was denatured and hybridized on a PSA-specific probe-coated microwell plate. Results. In 16 cancer cell lines, only LNCaP cells expressed especially high PSA mRNA values, with an optical density (OD) greater than 3. In other cell lines, two testicular cells had relatively high ODs, 1.909 and 0.987, respectively. A high PSA mRNA value was obtained by fine needle aspiration from pelvic lymph node specimens of cytologically positive lymph nodes from patients with prostate cancer but not from patients with cytologically proved bladder or penile cancer. Sensitivity and specificity of fine needle aspiration samples were 70% and 100%, respectively. Blood tests obtained from patients with prostate cancer demonstrated high PSA mRNA values. Conclusions. The PSA mRNA RT-PCR ELISA method provides a sensitive photometric enzyme immunoassay for the detection of PSA mRNA, using nonradioactive techniques.

Apoptosis in Proliferative Vitreoretinal Disorders: Possible Involvement of TGF-β-induced RPE cell Apoptosis

The targeted induction of apoptosis is a novel therapeutic approach to control the unlimited growth of proliferating cells. Since massive proliferation of cells at the vitreoretinal interface is a key feature of proliferative vitreoretinal disorders, we sought to identify apoptosis in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and macular pucker. Further, we evaluated the possible induction of apoptosis of retinal pigment epithelial (RPE) cells by transforming growth factor-β(TGF-β).Apoptotic cells were identified byin situDNA end labeling and acridine orange staining on paraffin-embedded tissue sections from epiretinal membranes of patients with all vitreoretinal disorders examined. Labeled nuclei or condensed chromatin were scattered throughout the membranes or occurred in clusters. Most apoptotic cells were RPE-derived, as assessed by cytokeratin immunochemistry. No apoptotic glial cells were detected. In PVR, proliferative activity, as confirmed by Ki-67 immunochemistry, was associated with short history and rapid disease progression. Apoptotic nuclei were observed more frequently in long-standing PVR or slow progression towards traction retinal detachment. TGF-βwas detected in all control vitreous samples by bioassay at concentrations below 20 ng ml-1. TGF-βlevels increased up to 20-fold in pathological vitreous. Marked heterogeneity was observed in all patient groups. The degree of TGF-βactivation was significantly higher in PVR than in PDR. Proapoptotic effects of TGF-βwere demonstrated in cultured human RPE cells by electron microscopy,in situDNA end labeling, comet assay and a photometric enzyme immunoassay for histone-associated DNA fragments.Apoptosis appears to be a key regulatory mechanism of growth control of specific cell populations in proliferative vitreoretinal disorders. Administration of proapoptotic growth factors such as TGF-βmay provide a novel approach to inhibit cellular proliferation at the vitreoretinal interface.

The "bystander effect": association of U-87 cell death with ganciclovir-mediated apoptosis of nearby cells and lack of effect in athymic mice.

Cells expressing the herpes simplex-thymidine kinase (HS-TK) gene as a consequence of retroviral transduction, as well as TK-negative (TK-) bystander cells, can be killed by treatment with ganciclovir (GCV). In vitro, this "bystander effect," has been attributed to metabolic cooperation through gap junctions or to the uptake of apoptotic vesicles. We show that GCV treatment kills TK-negative U-87 glioma cells cocultured with cells that express TK (TK+) but that have lost the capacity for releasing retroviral particles. A photometric enzyme immunoassay identifies histone-associated DNA fragments, typical of apoptosis, in the cytosol of GCV-treated TK+ cells, and apoptotic features are also demonstrated by ultrastructural studies. Northern blot analysis and the reverse transcription polymerase chain reaction (PCR) show that connexin 43, a major constituent of gap junctions, is expressed in TK+ and U-87 cells. The size of U-87 tumors in nude mice subsequently injected with TK+ cells and GCV is not significantly different than in untreated animals; whereas, after injecting 1:1 mixtures of U-87 and TK+ cells, GCV treatment only causes a temporary regression of tumor growth. On the contrary, when the injected mixtures contain PA317.STK.SBA (a retroviral producer cell line that can transduce efficiently the HS-TK gene) and U-87 cells, tumors are destroyed effectively by GCV treatment. Thus, an experimental setting in which U-87 gliomas are matched with cells that are able to express, but not to transduce, the HS-TK gene indicates that the bystander effect kills U-87 cells in vitro by mechanisms associated with apoptotic death. In vivo, this effect is not sufficient to restrain the tumor growth taking place in immunodeficient animals.