DNA Synthesis Of Target Cells Research Articles

Kupffer cells participate in 2-butoxyethanol-induced liver hemangiosarcomas

2-Butoxyethanol increases hemangiosarcomas selectively in male mouse liver after chronic inhalation through mechanisms that have not fully been elucidated. Hemolysis, a primary toxic effect associated with 2-butoxyethanol exposure in rodents, increased hemosiderin (iron) deposition in Kupffer cells in the liver. These findings, along with the induction of hepatic neoplastic lesions, led to our hypothesis that the induction hemangiosarcomas by 2-butoxyethanol is due to the activation of Kupffer cells, subsequent to hemolysis, that results in the induction of DNA synthesis in target cells (endothelial cells); allowing for the selective proliferation of preneoplastic target cells and/or the promotion of new initiated cells. The present studies were conducted to determine whether Kupffer cells contributed to 2-butoxyethanol-induced endothelial DNA synthesis in the liver, thereby determining whether a linkage exists between these events. Male B6C3F1 mice were treated with 450 and 900 mg/kg 2-butoxyethanol (via daily gavage; 5×/week) for 7 days in the presence or absence of Kupffer cell depletion (via clodronate-encapsulated liposomes). 2-Butoxyethanol (450 and 900 mg/kg/day) increased the number of F4/80 stained cells (Kupffer cells) compared to controls (∼1.3- and ∼1.6-fold over control, respectively). Clodronate liposome treatment reduced the number of Kupffer cells by >90%, as assessed by F4/80 immunohistochemistry. Increased hemolysis, measured by increases in relative spleen weights and decreased hematocrit was confirmed in 2-butoxyethanol treated mice. The percentage of iron-stained endothelial cells increased by ∼11-fold over control, and endothelial cell DNA synthesis increased ∼1.7-fold over control in 2-butoxyethanol exposed mice. Importantly, Kupffer cell depletion reduced 2-butoxyethanol-induced iron staining and hepatic endothelial cell DNA synthesis. These studies provide evidence supporting the hypothesis that the Kupffer cell modulates 2-butoxyethanol-induced endothelial cell DNA synthesis, and therefore may contribute to hemangiosarcoma induction by 2-butoxyethanol.

Engineering the NK1 Fragment of Hepatocyte Growth Factor/Scatter Factor as a MET Receptor Antagonist

The growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor MET, the tyrosine kinase encoded by the c-MET proto-oncogene, exert major roles in cancer invasion and metastasis and are key targets for therapy. NK1 is an alternative spliced variant of HGF/SF that consists of the N-terminal (N) and first kringle (K1) domains and has partial agonistic activity. NK1 crystallises as a head-to-tail dimer with an extensive inter-protomeric interface resulting from contacts between the two short interdomain linkers and reciprocal contacts between the N and K1 domains. Here we show that a subset of mutants at the NK1 dimer interface, such as the linker mutants Y124A or N127A or the kringle mutant V140A:I142A, bind the MET receptor with affinities comparable to wild-type NK1 but fail to assemble a dimeric, signalling competent NK1–MET complex. These NK1 variants have no detectable agonistic activity on, behave as bona fide receptor antagonists by blocking cell migration and DNA synthesis in target cells and have strong prospects as therapeutics for human cancer.

Enhancement of gene transfer with recombinant adeno-associated virus (rAAV) vectors into primary B-cell chronic lymphocytic leukemia cells by CpG-oligodeoxynucleotides

Objective Transduction of primary B-cell chronic lymphocytic leukemia (B-CLL) cells with recombinant adeno-associated virus (rAAV) vectors is dependent on preactivation of leukemic cells by CD40L. CpG-oligodeoxynucleotides (CpG-ODNs) are able to activate cytokine production and proliferation of B-CLL cells. Therefore CpG-ODNs were tested for their potential to enhance transgene expression in CLL cells. Materials and methods Using an optimized adenovirus-free packaging system, rAAV vectors coding for the enhanced green fluorescent protein (AAV/EGFP) were packaged and highly purified resulting in infectious titers up to 5×10 9/mL. Cells obtained from patients with B-CLL were infected with AAV/EGFP at a multiplicity of infection of 100 while being stimulated with CpG-ODNs and/or CD40L-expressing HeLa/SF cells. Transgene expression was assessed after 48 hours by flow cytometry. Results Stimulation of B-CLL cells by CpG-ODNs resulted in up-regulation of costimulatory molecules and G 1/S-phase transition at similar levels compared to activation by HeLa/SF cells, but use of CpG-ODNs alone did not result in any efficient AAV/EGFP transduction. Combined stimulation of B-CLL cells with HeLa/SF cells and CpG-ODNs during AAV/EGFP transduction significantly enhanced transgene expression compared to feeder stimulation alone ( p = 0.004). In addition, the copy number per single cell was significantly increased by addition of CpG-ODNs as detected by quantitative real-time PCR ( p = 0.04). Use of self-complementary AAV vectors that are not dependent on target cell DNA synthesis did not result in increased transgene expression compared to single-stranded AAV vectors ( p = 0.30). Conclusion Stimulation by CD40L is crucial for efficient gene transfer into B-CLL cells by rAAV vectors, whereas transduction efficiency can be significantly enhanced by CpG-ODNs.

Differential cytostatic effects of NO donors and NO producing cells

A 3-h exposure to NO donors (spermine-NO, DETA-NO, or SNAP), or to NOS II-expressing cells (activated macrophages or EMT6 cells) reversibly inhibited DNA synthesis in K562 tumor cells. In GSH-depleted K562 cells, cytostasis remained reversible when induced by DETA-NO or NOS II activity, but became irreversible after exposure to spermine-NO or SNAP. Only SNAP and spermine-NO efficiently inhibited GAPDH, an enzyme with a critical thiol, in GSH-depleted cells. Thus, the irreversible cytostasis induced in GSH-depleted cells by spermine-NO or SNAP can be tentatively attributed to S-nitrosating or oxidizing species derived from NO. However, these species did not contribute significantly to the early antiproliferative effects of macrophages. Ribonucleotide reductase, a key enzyme in DNA synthesis, has been shown to be inhibited by NO. Supplementation of the medium with deoxyribonucleosides to bypass RNR inhibition restored DNA synthesis in target cells exposed to DETA-NO and NO-producing cells, but was inefficient for GSH-depleted cells previously submitted to spermine-NO or SNAP. These cells also exhibited a persistent depletion of the dATP pool. In conclusion, GSH depletion reveals striking qualitative differences in the nature of the toxic effectors released by various NO sources, questioning the significance of S-nitrosating or oxidizing nitrogen oxides in NOS II-dependent cytostasis.

Amelioration of sodium barbital- induced nephropathy and regenerative tubular hayperplasia after a single injection of streptoxotocin does not abolish the renal tumor promoting effect of barbital sodium in male F344/NCr rats

The renal tumor promoting activity of barbital sodium (BBNa) and the role of renal tubular cell hyperplasia in tumor promotion following initiation with streptozotocin (STZ) was investigated. Six week old male F344/NCr rats were given STZ as a single i.p. injection of 50 mg/kg body wt and beginning 2 weeks later were fed diets containing 0 or 4000 p.p.m. of BBNa until they were killed at 33, 52 or 72 experimental weeks for histological evaluation and determination of levels of renal DNA synthesis by bromodeoxyuridine (Brdu) immunohistochemistry. A promoting effect on renal carcinogenesis was found by 72 weeks, but not at 33 or 52 weeks, confirming that prolonged administration of BBNa is necessary to promote renal tubular cell neoplasms. The promoting effect was evident as a higher incidence of large renal tubular tumors after 52 weeks, rather than an increase in number of dysplastic tubules, putative preneoplastic lesions. These findings suggest that the targets for the promoting activity of BBNa may be dysplastic lesions which may progress to tumors. Detailed examination by the step section technique through the entire kidneys revealed that STZ or BBNa administration induced a high incidence of putative preneoplastic renal tubular lesions (dysplasias) which seemed to be derived from the P1 or P2 segment of the nephron, also a site of high Brdu labeling index (LI) associated with BBNa toxicity. STZ administration was also associated with attenuation of BBNa-induced nephropathy and with diabetes from pancreatic islet degeneration and atrophy. The reduction in severity of nephropathy was correlated with a reduction in the LI of the renal cortical and medullary tubules, but not with the renal tumor incidence. These results indicate that decreased DNA synthesis in target cells does not eliminate tumor promoting activity in renal tubular epithelium. In addition, BBNa alone induced papillomas of the transitional epithelium of the renal pelvis and renal tubular cell adenomas.

Human monocyte-released cytotoxic factor: effect on various cellular functions, and dependency of cytolysis on various metabolic processes.

The ability of the human monocyte-released cytotoxic protein factor(s) (CF) to mediate cytolysis when various metabolic processes in target cells were inhibited, and its effect on various cell functions have been studied. Cytolysis was reduced when target cells were exposed to either chloroquine or colchicine, indicating that lysosome function and the formation of microtubule are of importance with respect to the interaction between CF and target cells, possibly by effecting internalization and processing CF and its receptor. A decrease in the energy charge of the target cells did not affect the ability of CF to mediate cytolysis, since the 60-70% reduction of the cellular ATP content by uncoupling oxidative phosphorylation had little effect on CF-induced lysis. It was unnecessary for target cells to be actively growing and dividing for CF to induce lysis because inhibition of cellular protein, RNA, and DNA synthesis potentiated the cytolytic effect of CF. CF-induced cell lysis of Walter and Eliza Hall Institute (WEHI) 164 target cells was first detected between 3.0 and 4.5 hr after addition of CF. Target cell DNA synthesis nearly terminated within 3-4 hr, about the time of first cell lysis detection. Compared to the effect on DNA synthesis, the CF effect on protein synthesis was negligible and only a slight reduction was detected in the RNA synthesis and in the cellular ATP content. The results indicated that the DNA-synthesizing machinery may either be a primary target site of CF or a rapid influence of CF action on its primary target site; however, neither the protein- or RNA-synthesizing machinery nor the cellular ATP generating systems are likely to be primary target sites.

Effect of Human Monocyte‐Released Cytostatic Factors on Target Cell DNA Synthesis, Protein Synthesis, and ATP Content

Cultured human monocytes activated in vitro with lymphokines and lipopolysaccharide release cytostatic protein factors that may be separated by ion-exchange chromatography into two populations, termed cytostatic factors I and II (CF I and II). The effect of CF I and II on target cell DNA synthesis, protein synthesis, and ATP content and the possible presence of a CF-associated protease activity were investigated. Inhibition of DNA synthesis was detected 4 h after addition of either CF I or CF II, whereas inhibition of protein synthesis was first detected after 10-15 h. Moreover, 20 h after addition of either CF I or CF II the average DNA synthesis per cell was inhibited by 25-35%, as compared with 10-15% inhibition of protein synthesis. No significant alteration in cellular ATP content was observed in cells culture up to 25 h with either CF I or CF II. Thus, neither protein synthesis nor generation of ATP appeared to be primary targets of either CF I or CF II, but the factors may act more directly on DNA synthesis. Protease activity was not associated with either CF I or CF II, and the factors thus differed from a previously reported cytolytic protease released from activated murine macrophages.

Soluble cytostatic factor(s) released from human monocytes. II. Effects on target cell kinetics.

Human monocytes release a stable cytostatic factor during in vitro culture after stimulation with lymphokine and endotoxin. The cell cycle time of synchronized NHIK 3025 cells increased from 20.3 to 23.2 h in the first cell cycle when the target cells were exposed to the factor during the whole cell cycle. In exponentially growing NHIK 3025 cell cultures the cell doubling time increased from 18.9 h to 23.1 h under continuous factor exposure for 70 h. These cells regained normal cell division rate when fresh culture medium replaced the cytostatic factor. Continuous exposure to the cytostatic factor for 96 h increased the cell doubling time of asynchronous K-562 cells from 19.7 to 31.8 h. The target cell DNA synthesis, evaluated by thymidine incorporation, was markedly depressed after culture with the factor for 24 h, and this depression was detectable already within 4 h of culture. The factor showed no cytolytic effect on the two cell lines tested. The cytostatic factor influenced the cell cycle distribution of both target cells tested, since cell cycle analysis by DNA flow cytometry demonstrated a reversible inhibition of factor-exposed NHIK 3025 cells in G1- and early S-phase, whereas K-562 cells accumulated in G1-phase.

Human monocytes induce a reversible inhibition of cell cycle progression in the leukemic cell line K-562 during co-culture in vitro.

Human monocytes cultured in vitro exert cytostatic activity against the non-adherent human leukemic cell line K-562. K-562 cells in asynchronous growth were added as target cells to adherent mononuclear blood cells, either freshly isolated or cultured in vitro for 4 and 12 days. After 24 hours of co-culture, the non-adherent cells were gently removed and incubated further. Target cell DNA-synthesis and cellular DNA content were examined by methyl-3H-thymidine incorporation and by flow cytofluorometry, respectively. Cell-proliferation curves were obtained. Monocytes cultured in vitro exert a reversible cytostatic activity, which is a partial cell cycle block in the G0/G1 phase. Target cells exposed to freshly isolated monocytes also showed accumulation of cells in the G0/G1 phase. These target cells did not regain their normal proliferation rate before day 3 after removal from the monocyte cultures. This effect may, however, to some extent be due to loosely adherent mononuclear blood cells removed from the monocyte cultures together with the target cells.

Tumoristatic effects of nonimmune BALB/c peritoneal macrophages on syngeneic lymphoma cells in vitro.

Peritoneal macrophages from unstimulated nonimmune BALB/c mice exerted nonspecific cytostatic effects of tumor cells in vitro. Incubation of syngeneic B40 lymphoma or allogeneic B16 melanoma cells with peritoneal macrophages or with macrophage culture supernatant fluids (SF) resulted in decreased target cell DNA synthesis and growth; in contrast, nontumorigenic syngeneic 3T3 fibroblasts were stimulated by macrophages but SF exerted no significant effects. Lipopolysaccharide activation of macrophages did not induce cytotoxicity for any target cell tested, and did not enhance the observed tumoristatic effects. The level of tumor inhibition associated with macrophage culture SF was dependent upon the concentration of fetal calf serum present in the medium as well as upon the numbers of macrophages cultured. Inhibitory SF could be harvested continuously from macrophage cultures for at least 7 days with no appreciable loss of activity.

Inhibition of DNA synthesis of target cells in vitro by sensitized lymphocytes.

SEVERAL experimental systems have been developed to test the cytotoxic effect of sensitized lymphocytes on target cells in vitro1–7. In a previous report8 we presented evidence for the inhibitory effect of lymphocytes on DNA synthesis in the target cells. Although this effect is strongest for the sensitized lymphocytes, the fact that it is also shown by the non-sensitized lymphoid cells casts some doubt as to its specificity. A further examination of this phenomenon was therefore undertaken.