Cross-linking of DNA induced by chloroethylnitrosourea is presented by O6-methylguanine-DNA methyltransferase.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase has been used as a reagent to analyse the initial reaction sites of alkylating agents such as chloroethylnitrosourea that cross-link DNA. The transferase can be employed for this purpose because it removes substituted ethyl groups from DNA, as shown by its ability to act on O6-hydroxyethylguanine residues in DNA. The enzyme counteracts the formation of interstrand cross-links induced by bis-chloroethylnitrosourea, but not those induced by nitrogen mustard. Once formed, chloroethylnitrosourea-induced cross-links are not broken by the enzyme. In agreement with deductions from experiments with living cells, it is concluded that chloroethylnitrosourea act by forming reactive monoadducts at the O6 position of guanine and/or the O4 position of thymine, which subsequently generate -CH2CH2- bridges to the complementary DNA strand. A new method for quantitating interstrand cross-links in DNA has been employed.     

The effect of various cytokines on the in vitro induction of antibody-dependent cellular cytotoxicity in murine cells. Enhancement of IL-2-induced antibody-dependent cellular cytotoxicity activity by IL-1 and tumor necrosis factor-alpha

We have previously demonstrated that incubation with IL-2 can induce ADCC activity in murine cells and that this activity was mediated by asialo GM1+, FcR+ cells. In the present study we show that the cytokines IFN-alpha and IFN-gamma, TNF-alpha, and IL-1 alpha are unable to induce antibody-dependent cellular cytotoxicity (ADCC) in murine cells; however, TNF-alpha and IL-1 alpha could substantially augment the ADCC induced by IL-2. IL-1 increased the IL-2-induced ADCC activity in a dose-dependent fashion and in cells isolated from the thymus and spleen. The precursors of the ADCC induced by the combination of IL-1 and IL-2 were asialo GM1+ cells, similar to the precursor cells of IL-2-induced ADCC. The effect of IL-1 and TNF on ADCC was not the result of an increase in the FcR density on the cell surface or the result of an increase in the number of FcR+ cells although IL-1 increased the recovery of viable cells in culture. The main effect of IL-1 and TNF was the enhancement of the lytic ability of the IL-2 cultured cells as indicated by increased intra-cellular benzyloxycarbonyl L-lysine thiobenzylester-esterase activity. These results suggest that lymphokines such as IL-1 and TNF may synergize with IL-2 in the induction of ADCC and could thus potentially be useful for the immunotherapy of established tumors when combined with the administration of specific anti-tumor antibodies.     

Enhancement of natural and antibody-dependent lymphocyte cytotoxicity by drugs which inhibit prostaglandin production by tumor target cells

Our previous observations suggested that the production of prostaglandins by tumor cells exposed to lymphocytes might constitute a mechanism by which the tumor cells Could subvert the effects of a cellular immune response directed against them. The present experiments tested this hypothesis by determining whether inhibition of prostaglandin production permitted enhanced expression of natural and antibody-dependent lymphocyte cytotoxicity against the target cells. Cell lines T₂₄ and HCV₂₉ were labelled with ⁵¹Chromium and incubated with purified lymphocytes obtained from venous blood of normal donors. Antiserum to T₂₄ and varying concentrations of inhibitors of prostaglandin synthetase (indomethacin, fenclozic acid, acetylsalicylic acid, and 2,6-xylenol) were added at the onset of incubation and assay tubes were incubated for varying times at 37 °C. In some experiments, lymphocytes or labeled target cells were preincubated with inhibitors and then washed prior to their addition to the assay tubes. Cytotoxicity was determined by measuring ⁵¹Chromium release and assessing any differences that might reflect the presence of the various drugs. Each prostaglandin synthetase inhibitor significantly enhanced both natural and antibody-dependent lymphocyte cytotoxicity. Enhancement appeared to reflect an effect on the target cells, presumeably by an inhibition of prostaglandin production. No increase in spontaneous ⁵¹Chromium release was apparent. The inhibitors did not appear to activate lymphocytes. This evidence supports the suggestion of a mechanism in which tumor cells may prevent the effect of a cellular immune response by producing inhibitory levels of prostaglandins. These results also suggest that manipulation of this mechanism can enhance the effectiveness of the lymphocyte response and may be a consideration in assessing lymphocyte/tumor cell interaction in vitro and in vivo.     

Slow cytotoxicity of the polysaccharide levan on tumor cells in vitro

Previous studies have shown that 1 h preincubation with levan caused a sharp decrease in tumorigenicity of tumor cells, although cell viability was not affected. In the present study, the possibility that levan might change the sensitivity of Lewis lung carcinoma cells to the host immune system and that levan might cause delayed damage were tested. Cell morphology, DNA synthesis, cell multiplication and viability as well as osmotic fragility were followed during several days in culture. Levan was found not to affect cell immunogenicity. The polysaccharide destroyed tumor cells by a rather peculiar mechanism: after a seemingly normal or even enhanced growth in culture during 3-5 days, cells suddenly burst. During the apparently normal growth, several morphological changes were observed: cell volume increased, cytoplasm swelled by apparent water uptake and some cells contained two or more nuclei. Levan-treated cells were found to be more susceptible to osmotic shock than non-treated cells. The reason for the sudden cell death could be a gradual increase in volume, up to a point which is no more compatible with membrane integrity, resulting in cell lysis.     

Selective cytotoxicity of scFv-TNF fusion protein on the tumor cells

A fusion protein of single-chain, Fv-tumor necrosis factor , scFv-TNF, was constructed and expressed in the Pichia pastoris expression system. There was 67-fold less toxicity of the fusion protein when compared with TNF- alone in cells expressing CA125. Furthermore, scFv-TNF was 10-fold more selective in cell killing directed by anti-CA125 scFv in a CA125-positive cell line than the CA125-negative line.     

Effect of lentinan on macrophage cytotoxicity against metastatic tumor cells

We studied the effect of lentinan, a fungal polysaccharide immunomodulator, on mouse peritoneal macrophages. The i.p. treatment of mice with 10 mg/kg lentinan affected the number, plastic-adherence, and endogen peroxidase activity of peritoneal cells. The cytotoxicity of lentinan-stimulated peritoneal macrophages was determined against several murine and human metastatic tumor targets: Lewis lung carcinoma (LLT) and two human melanomas, and was found to be significantly higher than that of the macrophages from control animals. However, the highly metastatic variant of LLT (LLT-HH) was resistant to the cytolytic effect of resident and lentinan-activated macrophages as well, indicating that the stimulation for cytotoxicity depends not only on the functional activity of the effector but also on the sensitivity of the target.     

Sensitizing effect of 3-methyladenine on radiation-induced cytotoxicity in radio-resistant HepG2 cells in vitro and in tumor xenografts

Many recent efforts have focused on targeting cell death pathways for discovering new cancer therapies. The relative resistance of liver cancer cells to ionizing radiation (IR) and chemotherapeutic agents due to autophagic response limits the available treatment options for this type of cancer. In this study, 3-methyladenine (3-MA), an autophagy inhibitor, was investigated for its potential to enhance radio-sensitivity under radio-resistant conditions both in vitro and in vivo. Hep3B and HepG2 cells were used to examine the radio-resistance of liver cancer cells. The results show that Hep3B cells respond to irradiation with increased apoptotic cell death and that HepG2 is radio-resistant due to the IR-induced autophagy, as verified by DNA fragmentation, electron microscopy, acidic vesicular organelle formation, and Western blot analysis. Application of IR with 3-MA to inhibit autophagy simultaneously suppressed the expression of LC3 and enhanced cell death. The tumor xenograft model in nude mice verified the synergistic cytotoxic effect of 3-MA and IR, which resulted in significant repression of tumor growth. The results demonstrate that IR-induced autophagy provides a self-protective mechanism against radiotherapy in HepG2 cells. In addition, 3-MA enhances the cytotoxicity of IR in cell models and suppresses tumor growth in animal models. Based on the results, application of 3-MA, or other autophagy inhibitors, could be used as an adjuvant for radiotherapy when radio-resistance develops as a result of autophagy response.     

Enhancement of cytotoxicity of artemisinins toward cancer cells by ferrous iron

Iron(II) heme-mediated activation of the peroxide bond of artemisinins is thought to generate the radical oxygen species responsible for their antimalarial activity. We analyzed the role of ferrous iron in the cytotoxicity of artemisinins toward tumor cells. Iron(II)-glycine sulfate (Ferrosanol) and transferrin increased the cytotoxicity of free artesunate, artesunate microencapsulated in maltosyl-beta-cyclodextrin, and artemisinin toward CCRF-CEM leukemia and U373 astrocytoma cells 1.5- to 10.3-fold compared with that of artemisinins applied without iron. Growth inhibition by artesunate and ferrous iron correlated with induction of apoptosis. Cell cycle perturbations by artesunate and ferrous iron were not observed. Treatment of p53 wild-type TK6 and p53 mutated WTK1 lymphoblastic cells showed that mutational status of the tumor suppressor p53 did not influence sensitivity to artesunate. The effect of ferrous iron and transferrin was reversed by monoclonal antibody RVS10 against the transferrin receptor (TfR), which competes with transferrin for binding to TfR. CCRF-CEM and U373 cells expressed TfR in 95 and 48% of the cell population, respectively, whereas TfR expression in peripheral mononuclear blood cells of four healthy donors was confined to 0.4-1.3%. This indicates that artemisinins plus ferrous iron may affect tumor cells more than normal cells. The IC(50) values for a series of eight different artemisinin derivatives in 60 cell lines of the U.S. National Cancer Institute were correlated with the microarray mRNA expression of 12 genes involved in iron uptake and metabolism by Kendall's tau test to identify iron-responsive cellular factors enhancing the activity of artemisinins. This pointed to mitochondrial aconitase and ceruloplasmin (ferroxidase).     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

Human monocyte cytotoxicity to tumor cells. I. Antibody-dependent cytotoxicity.

Recent investigations examining mononuclear cell antibody-dependent cell-mediated cytotoxicity against tumor cell lines suggest that K lymphocytes and not monocytes are active in this cytotoxic reaction. We have found, however, that in an allogeneic assay system, human monocyte monolayers as well as lymphocytes mediate substantial lysis of 51Cr-labeled antibody-coated CEM lymphoblast tumor cells. This cytotoxicity is temperature-dependent and rapid, with most 51Cr release occurring in the first 4 hr of co-incubation. Interaction between target cell-bound antibody and the monocyte Fc receptor is necessary as demonstrated by the marked fall in antibody-dependent cell-mediated cytotoxicity (ADCC) produced by staphylococcal protein A, high concentrations of nonspecific immunoglobulin, and dilution of the target cell antiserum. Morphologic and functional characteristics of the monocyte-monolayer preparations establish their relative purity (greater than 95%) and indicate that monocytes and not contaminating lymphocytes are responsible for tumor cell lysis. Furthermore, preincubation of monocyte and lymphocyte preparations with latex particles or low concentrations of immunoglobulin distinguished monocyte from lymphocyte ADCC. Thus, normal human monocytes have the capacity to carry out antibody-dependent cytotoxicity against nucleated malignant target cells.     

Pol kappa partially rescues MMR-dependent cytotoxicity of O6-methylguanine

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.     

Fc receptors on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.     

Human recombinant interleukin-6 enhances antibody-dependent cellular cytotoxicity of human tumor cells mediated by human peripheral blood mononuclear cells

Summary The effects of human recombinant interleukin-6 (hrIL-6) on antibody-dependent cellular cytotoxicity (ADCC) activity mediated by human peripheral blood mononuclear cells (PMNC) were investigated. Human PMNC were preincubated for 24 h with various concentrations of hrIL-6 and were used as effector cells in a 4-h⁵¹Cr-release assay. The ability of hrIL-6 to augment ADCC was measured using anti-colorectal carcinoma mAbs D612, 17.1A and 31.1 (each directed against a distinct tumor antigen) and using three human colorectal carcinoma cell lines, LS-174T, WiDr and HT-29, as targets. A significant increase in ADCC activity was observed after PMNC were preincubated in 100–400 U/ml but not in lower concentrations of hrIL-6. Variations in activities of PMNC among donors were observed. Non-specific mAb showed no effect in augmenting ADCC activity. hrIL-6 treatment did not augment non-specific (non-mAb-mediated) cytotoxicity. The enhancement of ADCC activity was blocked by the addition of an antibody against hrIL-6 but not by an antibody to the IL-2 receptor (capable of blocking the induction of lymphokine-activated killer cell cytotoxicity by IL-2), suggesting that hrIL-6 augmentation of ADCC activity may not be mediated through IL-2. These results demonstrate that hrIL-6 augments ADCC activity of human PMNC using mAbs to human tumor antigens and human tumor cells as targets, suggesting a potential role for IL-6 in combination with anti-cancer antibodies for cancer immunotherapy.     

Enhancement of radiation cytotoxicity by UCN-01 in non-small cell lung carcinoma cells

Thoracic ionizing radiation is a standard component of combined-modality therapy for locally advanced non-small cell lung cancer. To improve low 5-year survival rates (5- 15%), new strategies for enhancing the effectiveness of ionizing radiation are needed. The kinase inhibitor UCN-01 has multiple cell cycle effects, including abrogation of DNA damage-induced S- and G(2)-phase arrest, which may limit DNA repair prior to mitosis. To test the hypothesis that therapy-induced cell cycle effects would have an impact on the efficacy of a combination of UCN-01 plus ionizing radiation, the cell cycle responses of the non-small cell lung cancer cell lines Calu1 (TP53-null) and A549 (wild-type TP53) to 2 Gy ionizing radiation were correlated with clonogenic survival after irradiation plus UCN-01. Irradiated cells were exposed to UCN-01 simultaneously and at 3-h increments after irradiation. In Calu1 cells but not A549 cells, sequence-dependent potentiation of radiation by UCN-01 was observed, with maximal interaction occurring when UCN-01 was administered 6 h after irradiation. This coincided with the postirradiation time with the greatest depletion of cells from G(1). Abrogation of G(2) arrest was observed regardless of TP53 status. The role of TP53 was investigated using siRNA to achieve gene silencing. These studies demonstrated that radiation plus UCN-01 was more effective in cells with diminished TP53 activity, associated with a reduced G(1) checkpoint arrest. These studies indicate that simultaneous elimination of multiple DNA damage-induced checkpoints in G(1), S and G(2) may enhance the effects of radiation and that drug scheduling may have an impact on clinical efficacy.     

Lipid vesicle interaction with EMT-6 tumor cells and effect on subsequent cell growth

Artificial lipid vesicles of varying composition were incubated with EMT-6 tumor cells, after which the transfer of vesicle lipids to the cells and their growth $\text{in}\text{vitro}$ were determined. Vesicles composed of phosphatidylcholine, cholesterol, and gangliosides could transfer phosphatidylcholine and cholesterol to the cells, but this transfer had no effect on the subsequent growth of the cells. However, preincubation of the cells with vesicles containing sterylamine or phosphatidylserine did inhibit the subsequent growth of the cells in a tissue culture assay system. It might be possible to deliver to certain cells growth inhibitory compounds carried in lipid vesicles.     

Cytostatic effect of immune system cells on tumor cells

The cytostatic effect of different lymphoid cells on tumour cells was studied in mice. It was shown that the cells differed in their ability to manifest cytostatic and natural cytotoxic activity. The interstrain variations of cytostatic activity levels in murine splenocytes were revealed. The degree of cytostatic action of effector cells on tumour cells depended both on effector-target cells ratio and on the incubation time of effector and target cells. The cytostatic effect of splenocytes and macrophages was demonstrated to be unrestricted by H-2 complex and independent of the tumour type. The data suggest that the cytostatic effector cells are heterogeneous and may be distinct from natural killer cells.     

Enhancement of misonidazole cytotoxicity by iron

The toxicity of misonidazole (MISO) to hypoxic Chinese hamster ovary (CHO) cells in serum-free medium is enhanced by Fe(III)-EDTA. Enhancement of MISO cytotoxicity by a factor of 1.6 was seen with 2 microM Fe(III)-EDTA, while 200 microM Fe(III)-EDTA results in sensitization by a factor of 2.0. Treatment of CHO cells with the iron chelator desferal resulted in protection against the hypoxic cytotoxicity in MISO (approximate protection factor of 2.5 with 100 microM desferal). Similar results were obtained with Chinese hamster V79 cells. Fe(III)-EDTA also enhanced binding of [2-14C] MISO to cellular macromolecules while desferal decreased binding of MISO to cellular macromolecules. These results suggest that iron plays an important role in the reductive metabolism of MISO and that modification of the intracellular metal ion status may be a useful approach to modulating the biological effect of nitro compounds.