The molecular mechanisms of 5-fluoro-2'-deoxyuridine induced cell death

The molecular mechanism of cell death induced by 5-Fluoro-2'-deoxyuridine (FUdR) was investigated. FUdR caused cell death to induce dNTP pool imbalance and following DNA double strand breaks in mouse mammary tumor FM3A cells. We isolated a new endonuclease from FUdR-treated cells, named endonuclease S, that played an important role in FUdR-induced cell death. Cells treated with FUdR showed intracellular acidification before cell death formation. We observed that the endonuclease S in acidic cells may lead the DNA fragmentation. On the other hand, we observed that protease inhibitors (such as TLCK, TPCK, PMSF, p-APMSF, Pefabloc SC and Z-Asp-CH2-DCB) blocked intracellular acidification, DNA fragmentation and FUdR-induced cell death. But the inhibitors did not affect dNTP pool imbalance in the cells. These results suggest that proteases act at the point of downstream of dNTP pool imbalance and upstream of the intracellular acidification.     

Induction of programmed death/apoptosis androgen-dependent mouse mammary tumor cell line (Shionogi Carcinoma 115) by androgen withdrawal

Shionogi Carcinoma 115 (SC 115) cells are a cloned cell line derived from androgen-dependent mouse mammary tumor. They can grow in serum-free culture if a physiological level of androgen is present in the medium, but can not proliferate in culture without testosterone. In the present study, the mechanism of cell death in SC 115 cells after androgen withdrawal was examined. Based upon the temporal sequence of DNA fragmentation, morphologic changes and loss of cell viability, androgen withdrawal induces programmed cell death (apoptosis) of SC 115 cells in serum-free culture. Northern blot analysis was used to identify a series of genes whose expression per cell is enhanced during the recruitment of cells from a nonproliferative (i.e. G0) state into G1 (i.e.,cyclins D1 and C), from G1 into the S phase of the cell cycle (i.e., cdk2), and during the programmed cell death pathway (i.e. testosterone repressed prostatic message-2 (TRPM-2), transforming growth factor-beta1 (TGF-beta1) and glucose regulated 78 kilodalton protein (GRP-78). Expression of TRPM-2, TGF-beta1, GRP-78, and calmodulin genes increases, but that of cyclins C and D1, and cdk2 genes decreases during programmed cell death of SC 115 cells. These results demonstrate that androgen-dependent SC 115 cells undergo programmed cell death induced by androgen withdrawal, and that this death does not require proliferation or progression into G1 of the proliferative cell cycle. SC 115 cells should be a good model for investigating programmed death of hormone-dependent cancer.     

Conjugation of 5-fluoro-2''-deoxyuridine with lactosaminated poly-l-lysine to reduce extrahepatic toxicity in the treatment of hepatocarcinomas

Numerous clinical results reported in the literature prove that ceramic femoral heads reduce wear, osteolysis, and revision rates in total hip replacements (THRs). Femoral heads and cups made out of alumina ceramics were introduced about 25 years ago, and femoral heads made out of zirconia were introduced about 10 years ago. Today, the wear couples of alumina-on-polyethylene or zirconia-on-polyethylene have become the standard in THR. The wear couple offering the lowest wear rate is alumina-on-alumina. This article discusses the state of the art and lists advantages as well as problems with the various materials used for prostheses in THR.     

The characterization of cell death induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl) cytosine (ECyd) in FM3A cells

The characterization of cell death induced by 1-(3-C-ethynyl-beta-D- ribopentofuranosyl) cytosine (ECyd), a potent inhibitor of RNA synthesis, was performed using mouse mammary tumor FM3A cells in vitro. Accompanied with the cell death induced by ECyd (3.0 muM) -treatment, about 100-200 kbp-sized and internucleosomal DNA fragmentation were observed by orthogonal-field-alternation gel electrophoresis (OFAGE) and conventional gel electrophoresis, respectively. Protease inhibitors, carbobenzoxy-L-aspart-1-yl[(2,6-dichlorobenzyl)oxy]methane (Z-Asp-CH2-DCB), N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), effectively blocked the cell death, suggesting that the proteases inhibited by Z-Asp-CH2-DCB, TLCK or TPCK were involved in the process of cell death.     

Apoptosis and necrosis: mechanisms of cell death induced by cyclosporine A in a renal proximal tubular cell line

BACKGROUND: The mechanisms of cyclosporine (CsA)-induced nephrotoxicity are not fully understood. While hemodynamic changes may be involved in vivo, there is also some evidence for tubular involvement. We previously showed direct toxicity of CsA in the LLC-PK1 renal tubular cell line. In the current study we examined mechanisms (apoptosis or necrosis) of cell death induced by CsA in the LLC-PK1 renal proximal tubular cell line. The possible role of the Fas (APO-1/CD95) antigen-Fas ligand system in the mediation of CsA-induced cell death was also investigated. METHODS: Cells were treated with CsA (0.42 nM to 83 microM) for 24 hours and alterations in DNA and protein synthesis and membrane integrity were examined. Flow cytometry was used to investigate: (i) alterations in the DNA content and cell cycle; (ii) the forward (FSC) and side (SSC) light scattering properties (indicators of cell size and granularity, respectively); (iii) the externalization of phosphatidylserine (PS) as a marker of early apoptosis using FITC-annexin V binding; and (iv) expression of the apoptotic Fas protein. DNA fragmentation in apoptotic cells was also determined by the TUNEL assay. RESULTS: CsA (all doses) caused a block in the G0/G1 phase of the cell cycle as indicated by a decrease in DNA synthesis and supported by an increase in the % of cells in the G0/G1 phase with concurrent decreases of those in the S and G2/M phases. The effect on protein synthesis appeared to be much less. Lower doses of CsA (4.2 nM) caused the appearance of a "sub-G0/G1" peak, indicative of reduced DNA content, on the DNA histogram that was paralleled by a reduction in cell size and an increased cell granularity and an increase in FITC-annexin V binding. DNA fragmentation was evident in these cells as assessed using the TUNEL assay. Higher doses of CsA increased cell size and decreased cell granularity and reduced membrane integrity. Expression of Fas, the cell surface molecule that stimulates apoptosis, was increased following low dose CsA exposure. CONCLUSIONS: These results indicate that CsA is directly toxic to LLC-PK1 cells with reduced DNA synthesis and cell cycle blockade. The mode of cell death, namely apoptosis or necrosis, is dose dependent. Fas may be an important mediator of CsA induced apoptosis in renal proximal tubular cells.     

Growth of stable clones of mouse fibroblast cell line C3H10T1/2 expressing the human retinoblastoma gene product

Phosphorylation and dephosphorylation of the retinoblastoma gene product (pRB) are recognized as necessary events in the cell cycle progression. To study the role of pRB in regulation of cell proliferation, the stable cell lines with constitutive expression of the exogenous RB gene can be employed. In order to obtain such cell lines in this work C3H10T1/2 mouse fibroblasts were infected with defective retrovirus encompassing the RB and Neo gene conferring resistance to geniticine (G418). The pRB production and its phosphorylation pattern were analyzed by immunoblotting in cell lysates considering well known data on correlation between pRB phosphorylation pattern and its electrophoretic mobility. Cell lines subjected to G418 selection with the following cloning procedure were identical to the control cells expressing beta-galactosidase, when compared for pRB production and phosphorylation in the cell cycle stages characterized by hyperphosphorylated pRB. However, cells of the experimental cell lines hypophosphorylated pRB much faster and accumulated much more underphosphorylated protein compared to the control cell lines. The doubling time of the cells was not affected either by changes in the pRB phosphorylation pattern or by its overproduction during separate cell cycle stages. These results suggest that maintaining of the physiological level of pRB phosphorylation in cycling cells is strictly controlled and is considered to be a more important condition of the cell cycle progression than pRB dephosphorylation.     

Induction of antitumor immunity by interleukin-2 gene-transduced mouse mammary tumor cells versus transduced mammary stromal fibroblasts

BACKGROUND: Tumor cell-targeted cytokine gene transfer has been used to generate tumor cell vaccines, but this approach is limited by the need to establish and implant live tumor cells. PURPOSE: The purpose of this study was to determine if stromal fibroblasts could be used as an alternative vehicle for delivery of the cytokine interleukin-2 (IL-2) into the tumor microenvironment. We attempted to establish the feasibility of (a) genetic immunotherapy in a mammary tumor system and (b) engineering stromal fibroblasts as well as tumor cells. We compared the effects of tumor cell-mediated and stromal fibroblast-mediated local IL-2 expression on the generation of antitumor immune responses. METHODS: Retroviral vectors containing a human IL-2 gene were used to transduce a mouse mammary tumor line, 4TO7, and an immortalized but nontumorigenic fibroblast line established from syngeneic mammary fatpads. Expression of the IL-2 gene in transduced cells was determined by measuring IL-2 secretion, by RNA-polymerase chain reaction, and by immunochemistry. Groups of 5-12 BALB/c mice were injected with either 4TO7 cells or various doses of IL-2-secreting 4TO7 cells (4TO7-IL-2); tumor growth was monitored. To test whether local IL-2 expression by transduced cells could influence the growth of unmodified tumor cells, we determined tumor development in groups of mice treated with 4TO7 cells co-injected with either 4TO7-IL-2 cells or IL-2-secreting fibroblasts. RESULTS: 4TO7-IL-2 cells induced active immunity able to reject the immunizing tumor and to resist challenge with parental 4TO7 cells on the contralateral side. Mice pretreated with 4TO7-IL-2 were significantly protected compared with untreated control animals or mice pretreated with irradiated 4TO7 cells. The immunity induced by 4TO7-IL-2 cells did not protect against challenge with another subline, 4T1, which was derived from the same spontaneously arising mammary tumor as 4TO7. Co-injection of 4TO7 cells with 4TO7-IL-2 cells reduced tumorigenicity, whereas co-injection of 4TO7 cells with IL-2 secreting fibroblasts did not. CONCLUSION: Our results suggest that induction of anti-tumor immune response by local IL-2 production is most effective when the helper cytokine is secreted by the tumor cell. IMPLICATION: Our studies caution against the use of IL-2 gene-transduced syngeneic stromal cells as an alternative strategy of gene therapy for cancer. However, they may allow study of the mechanisms of tumor antigen recognition and the possible involvement of co-stimulatory signals for effective tumor vaccination by gene-modified cells.     

Toxicity of liposomal 3'-5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine in mice

Toxicities of 5-fluoro-2'-deoxyuridine (FUdR) and its liposome incorporated dipalmitoyl derivative (FUdR-dipalmitate) to mouse bone marrow, spleen, liver and ileum were compared after treatment for 6 consecutive days. The applied doses of the two formulations, which were shown earlier to have equal antitumor activity in mouse tumor models, were 600 and 2 mumol/kg respectively. When applied in these doses, toxicity to the hemopoietic system, measured as a decreases in progenitor and precursor cells of the erythroid and granuloid/macrophage lineage in bone marrow and spleen, was more severe for FUdR than for liposomal FUdR-dipalmitate. In the liver, mitotic figures, as indicators of cell division, were absent for both drugs while in control livers the number of cells in mitosis was approximately 2%. Toxicity to the ileum was more severe for liposomal FUdR-dipalmitate than for FUdR and was manifested by granulocyte infiltration, the presence of cell debris, loss of columnar epithelial cells and enlarged nuclei with prominent nucleoli in these cells. Thus, by prolonging the retention time of FUdR in vivo, using liposomes as a vehicle and FUdR-dipalmitate as a lipophilic prodrug, the dose-limiting toxicity appears to shift from bone marrow to the gastrointestinal tract.     

Analysis of mutations in the thymidine kinase gene of varicella zoster virus associated with resistance to 5-iodo-2''-deoxyuridine and 5-bromo-2''-deoxyuridine

The activity of monoamine oxidase (MAO) and dopamine-beta-hydroxylase (DBH), enzymes involved in monoamine metabolism, were studied in 29 bipolar patients (mean age = 33.12 years, SD = 7.27) who were treated with lithium carbonate and in 20 healthy volunteers (mean age = 30.05 years, SD = 6.04). Platelet MAO activity was higher after lithium withdrawal, whereas plasma DBH activity was lower in remitted euthymic bipolar patients compared with normal volunteers. During lithium treatment, platelet MAO activity decreased and plasma DBH activity increased compared with the lithium-withdrawal values. It was also observed that the activities of these enzymes in the bipolar patients during lithium treatment did not differ from those in the volunteers. Thus, platelet MAO and plasma DBH activities differed in unmedicated patients with bipolar affective disorder from those of healthy subjects. Treatment with lithium appeared to have a normalizing effect on MAO and DBH activity levels.     

Characterization of organ-specific immunoliposomes for delivery of 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine in a mouse lung-metastasis model

A previous study has shown that lipophilic prodrugs can be delivered efficiently to normal lung endothelium by incorporation into liposomes covalently conjugated to monoclonal antibody (mAb) 34A against the lung endothelial anticoagulant protein thrombomodulin. In the present study, the potential use of these lung-targeted immunoliposomes (34A-liposomes) for delivery of a lipophilic prodrug, 3',5'-O-dipalmitoyl-5-fluoro-2'-deoxyuridine (dpFUdR), to the tumor-bearing lung was examined using BALB/c mice bearing experimental lung metastasis induced by i.v. injection of EMT-6 mouse mammary tumor cells. Immunohistochemical examination of the tumor-bearing lung showed specificity of mAb 34A to lung endothelium. Tumor cells appeared to localize just outside of the normal blood vessels and were within a small diffusion distance from the mAb 34A-binding sites. 111In-labeled 34A-liposomes containing monosialoganglioside (GM1) were prepared that included [3H]-dpFUdR at 3.0 mol% in the lipid mixture. In vitro cell binding studies further demonstrated that 34A-liposomes bound specifically to normal mouse lung cells that expressed thrombomodulin but not to EMT-6 cells. Biodistribution study showed efficient and immunospecific accumulation of [3H]-dpFUdR incorporated into 34A-liposomes in the lung at a level parallel with that of 111In-labeled 34A-liposomes, indicating that the drug is delivered to the target organ in intact liposomes. Liposomal dpFUdR appeared to be metabolized in the lung to the parent drug FUdR at a rate slower than in the liver and spleen. Furthermore, treatment of lung-metastasis-bearing mice with dpFUdR incorporated into 34A-liposomes on days 1 and 3 after tumor cell injection resulted in a significant increase in the median survival time of treated mice as compared with control mice (%T/C value, 165%). dpFUdR either dispersed in emulsion or incorporated into antibody-free liposomes was ineffective in prolonging the survival of mice. These results indicate the potential effectiveness of organ-specific immunoliposomes containing a lipophilic prodrug for the targeted therapy of metastatic tumors.     

Isolation of a novel mouse gene MA-3 that is induced upon programmed cell death

Haematopoietic progenitor cells were isolated from human fetal liver, obtained between 6 and 15 weeks gestation. After preparation of a single cell suspension, the cells were stored using a stepwise freezing protocol; taking the cells from room temperature through -70 degrees C to liquid nitrogen. Viability (trypan blue exclusion), morphology (Leishman stain), identification of cell type (flow cytometry) and growth characteristics in semi-solid culture medium were assessed using the fresh cell suspension. We were able to confirm that the predominant cells in human fetal liver up to about 15 weeks gestation are those of the erythroid lineage. It was established that viability in excess of 75% was required to ensure adequate growth in culture after frozen storage and it was deemed important to ensure morphological integrity of the cell preparations. The colonies formed in culture were observed to be producing haemoglobin between 7 and 9 days after initial seeding. We have determined that cells can be stored in liquid nitrogen for up to 2 years without loss of (1) viability, (2) morphological features and (3) ability to form colonies and produce haemoglobin in culture. These findings offer encouragement for the implementation of a cell bank to support an in utero transplantation programme.     

A comparison of distinct modes of tumor cell death in Hodgkin's disease using morphology and in situ DNA fragmentation

The study examined the morphology and frequency of cell death occurring spontaneously in lymph nodes from patients with Hodgkin's disease. In addition to necrosis, which was infrequent and usually in patches, we document two cell types showing features of individual cell death: mummy cells end apoptotic cells. Mummy cells present no evidence of DNA fragmentation, but show electron microscopic features of "dark cells." Apoptotic Hodgkin-Reed-Sternberg cells are found frequently and are easier to demonstrate by in situ and labeling of fragmented DNA than by light microscopy only. In many cases phagocytosis of apoptotic cells is also documented. The significance of these findings to the limited number of Hodgkin-Reed-Sternberg cells in most cases of Hodgkin's disease is discussed.     

Isolation and characterization of a spontaneously transformed malignant mouse mammary epithelial cell line in culture

A method is described that permits the selection of spontaneously transformed mammary epithelial colonies from an untransformed mouse mammary epithelial cell line, NMuMG, and utilizes a long-term anchorage-independent growth of the transformants on soft agarose. These transformed cells (NMuMG-ST) are shown to be distinguishable from the untransformed cells by morphology, growth characteristics, induced carcinomas when transplanted into nude mice and ability to metastasize. This transformed phenotype displayed focal, multilayer growth and higher saturation density in comparison with the untransformed phenotype. Transplanted tumors as well as metastatic lung tumors in nude mice were adenocarcinomas morphologically similar to typical mammary tumors in humans. This selection procedure of mutant mammary cells from an immortalized cell line derived from normal mammary glands could be very useful to identify the genomic biomarkers in the growth regulation and malignant progression of breast cancer.     

Insulin binding and fluid-phase endocytosis stimulation in the mouse neuroblastoma cell line 41A3

As well as many other hormones and growth factors, insulin is known to influence several processes in the CNS; its specific effects, however, are still poorly understood. Neuroblastoma cell lines represent a useful experimental system for the analysis of the insulin-specific effect on neurons, in the absence of possible regulatory mechanisms elicited by other neuronal/glial cells and/or soluble factors. The expression and the binding properties of insulin receptors, as well as the insulin effects on both membrane fluidity and cell surface architecture, have been investigated in 41A3 mouse neuroblastoma cells, by radioligand-binding fluorescence spectroscopy and scanning electron microscopy, respectively the same cells, insulin-induced modifications on cytoskeletal organisation also have been studied. Binding studies were performed using 125I-insulin, while the cationic fluorescent probe trimethylammonium 1,6-diphenyl-1,3,5-hexatriene was used for biophysical investigations. The results presented in this paper provide evidence that insulin interacts with 41A3 neuroblastoma cells through a receptor-mediated mechanism and that, in these cells, insulin binding modifies the cell surface morphology and stimulates endocytosis.     

Characterization of the cell death process induced by staurosporine in human neuroblastoma cell lines

Staurosporine is a potent and non-specific inhibitor of protein kinases. There is also evidence of staurosporine being a potent inducer of apoptosis. In several human neuroblastoma cell lines (SH-SY5Y, NB69, IMR-5 and IMR-32) we have found 100 nM staurosporine to induce cell death in half the population (EC50). Electron microscopy of these cells, fluorescence microscopy after Hoechst-33258 staining of chromatin and agarose-electrophoresis of DNA, show two different types of cell death. SH-SY5Y and NB69 die by apoptosis and display all the characteristic features of it. IMR-5 and IMR-32 lack some of these features and a ladder pattern of DNA degradation is not found. Different morphological types of apoptosis have been described during the development of vertebrates; the possibility of finding a similar diversity in cell culture is suggested. On the other hand, staurosporine is a potent promoter of neurite outgrowth. In all the neuroblastoma cell lines we have tested, neurite-promoting and cell death-inducing staurosporine concentrations mostly overlap. This fact has not been reported before, probably because of an early versus late timing of these two different phenomena. The neuritogenic effect has prompted the suggestion that staurosporine could be a prototype of drugs for neurodegenerative diseases; the present study raises several concerns about such a proposal.     

Skin carcinogenesis induced be neonatal administration of 5-bromo-2''-deoxyuridine and subsequent treatment with 12-O-tetradecanoylphorbol-13-acetate in mice

A 75-year-old woman with severe rheumatic mitral stenosis was admitted for percutaneous mitral valvuloplasty. Two-dimensional echocardiography disclosed severe calcification of the posterolateral left atrial wall and interatrial septum. Percutaneous mitral valvuloplasty, using the inoue technique, was successfully performed after a transseptal puncture through the calcified interatrial septum, avoiding the need for surgical intervention.