Predominant suppression of neutrophil colony growth by recombinant human tumor necrosis factor

To investigate the suppressive effect of recombinant human tumor necrosis factor (rH-TNF) on colony growth of human granulocyte-macrophage progenitor cells (CFU-GM), cytochemical examinations of CFU-GM colonies were performed by a triple staining method. Each colony was classified into five subtypes, and the effects of rH-TNF on each subtype were analyzed. Neutrophil colony growth was inhibited by rH-TNF in a dose-dependent manner, and it was almost completely suppressed at 100 U/ml. In contrast, no significant suppressive effect of rH-TNF was found on the growth of monocyte-macrophage and eosinophil colonies at 100 U/ml or less. When recombinant human granulocyte colony-stimulating factor which almost exclusively stimulates neutrophil colony formation was used as a source of colony-stimulating activity, the total colony growth was almost completely suppressed by 100 U/ml of rH-TNF. These results indicate predominant inhibition of neutrophil colony growth by rH-TNF.     

Stimulation of uptake of tritiated thymidine into mouse marrow cells in culture by a factor from L-cell conditioned medium

Uptake of ³H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of ³H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The ³H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, ³H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.     

An extract of seeds fromAeginetia indica L., a parasitic plant,induces potent antigen-specific antitumor immunity in Meth A-bearing BALB/c mice

Summary The antitumor activity of an extract of seeds fromAeginetia indica L., a parasitic plant, was investigated. BALB/c mice, inoculated i.p. 1 × 10⁵ syngeneic Meth A tumor cells, were administered 2.5 mg/kgA. indica extract i.p. every 2 days from day 0. The untreated mice died of an ascitic form of tumor growth within 21 days, whereas all the treated mice completely recovered from tumor challenge without any side-effects. The extract did not exert direct cytotoxic activity against Meth A in vitro. Mice that survived after the first challenge as a result ofA. indica treatment overcame the rechallenge with homologous Meth A without additional administration of the extract. On the other hand, those mice could not survive after rechallenge with Meth 1 tumor cells, which were also established in BALB/c mice but were different in antigenicity from Meth A, suggesting the development of antigen-specific concomitant immunity in theA. indica-cured mice. In the induction phase of antitumor resistance in this system, CD4⁺ T cells appeared to be the main contributors, since in vivo administration of anti-CD4 mAb completely abolished such resistance. In contrast, anti-CD8 mAb administration did not influence the effect ofA. indica. The importance of CD4⁺ T cells in antitumor immunity was again clarified by Winn assay; that is, spleen and lymph node cells depleted of CD4⁺ T cells in vitro prior to assay abolished antitumor activity on co-grafted Meth A tumor cells in vivo.     

The effect of photosensitizing dyes on the 3H-thymidine incorporation of cells grown in vitro

The photodynamic inactivation of ³H-thymidine incorporation in mouse embryo (ME) and mouse L cells by acridine orange (AO), methylene blue (MB) or neutral red (NR) has been studied by estimating the number of nuclei capable of incorporating ³H-thymidine during a 24 h period following light exposure. In the dark NR and AO reduced the number of ME-nuclei incorporating ³H-thymidine but MB caused an increase in non-scheduled DNA synthesis. The dark effect on L cells was less but the photoinactivation of thymidine uptake was proportionally greater in these cells. Polyoma virus was shown to be capable of growing in cells whose thymidine uptake was reduced or completely stopped by photoinactivation with NR. However, if the NR damage was very great, or when AO was used to photosensitize cells, the synthesis of viral DNA was interfered with.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

A humanized anti-osteopontin antibody inhibits breast cancer growth and metastasis in vivo

Osteopontin (OPN) has been implicated as an important mediator of breast cancer progression and metastasis and has been investigated for use as a potential therapeutic target in the treatment of breast cancer. However, the in vivo antitumor effect of anti-OPN antibodies on breast cancer has not been reported. In this study, a mouse anti-human OPN antibody (1A12) was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. A humanized version of 1A12, denoted as hu1A12, was shown to possess affinity comparable to that of its parental antibody. The ability of hu1A12 to inhibit cell migration, adhesion, invasion and colony formation was assessed in a highly metastatic human breast cancer cell line MDA-MB-435S. The results indicated that hu1A12 was effective in inhibiting the cell adhesion, migration, invasion and colony formation of MDA-MB-435S cells in vitro. hu1A12 also showed significant efficacy in suppressing primary tumor growth and spontaneous metastasis in a mouse lung metastasis model of human breast cancer. The specific epitope recognized by hu1A12 was identified to be ²¹²NAPSD²¹⁶, adjacent to the calcium binding domain of OPN. Our data strongly support that OPN is a potential target for the antibody-based therapies of breast cancer. The humanized anti-OPN antibody hu1A12 may be a promising therapeutic agent for the treatment of human breast cancer.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Induction of deoxyribonucleic Acid synthesis in potato tuber slices: role of protein synthesis

Timing of protein synthesis which is a prerequisite to DNA synthesis induced in potato tuber tissue (Solanum tuberosum L.) by cut injury has been studied using cycloheximide. The induction of DNA synthesis which was measured by incorporation of ³H-thymidine was completely inhibited when the inhibitor was applied to the tuber discs immediately after slicing. When the application of cycloheximide was delayed for 6 hours or more after slicing, DNA synthesis was observed but its rate was reduced to 20% of control. The inhibitory effect of cycloheximide, however, rapidly decreased when the inhibitor was applied at 6 or less hours immediately prior to determination of DNA synthesis. The effect of cycloheximide on the incorporation of ¹⁴C-leucine suggests that the change in the effect of cycloheximide on the induction of DNA synthesis is not due to incomplete inhibition of protein synthesis. Cycloheximide did not have significant effects on either uptake or phosphorylation of ³H-thymidine in the discs. Inhibition of both protein and DNA synthesis by cycloheximide was reversed by washing and further incubation of the discs. Almost no qualitative difference was detected by buoyant density analysis between DNA formed under inhibition of protein synthesis of the later stage and DNA synthesized under normal conditions. These results suggest that DNA synthesis induced in potato tuber tissue by cut injury requires continuous synthesis of new protein molecules in a characteristically programmed sequence.     

Concanavalin A-stimulated Ca2+ uptake in rat splenocytes

Summary Commercially available concanavalin A binds Ca²⁺ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca²⁺ uptake (defined as an increased association of ⁴⁵Ca²⁺ with cells) in rat splenocytes and Ca²⁺ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in ⁴⁵Ca²⁺ uptake over control. The concanavalin A stimulated uptake of ⁴⁵Ca²⁺ occurred within minutes, and required concentrations of concanavalin A which promoted [³H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca²⁺ uptake than concanavalin A. Sodium periodate inhibited Ca²⁺ uptake at concentrations which promoted ³H-thymidine incorporation into splenocytes.It is concluded that con canavalin A promotes Ca²⁺ uptake which is not due to binding of ⁴⁵Ca²⁺ to concanavalin A. Although the concanavalin A-promoted Ca²⁺ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by ³H-thymidine incorporation, the role of Ca²⁺ in this event remains unclear.     

Cytokine effects and role of adhesive proteins and Fc receptors in human macrophage-mediated antibody dependent cellular cytotoxicity.

Mononuclear phagocytes participate in host immunological defense against tumors. We have investigated the role of selected recombinant cytokines on human macrophage-mediated tumor cytotoxicity in vitro utilizing a human colon cancer cell line target, SW1116, and murine monoclonal antibody 17-1A. Blood monocytes were kept in continuous culture to allow differentiation into macrophages. Maximum antibody dependent cellular cytotoxicity (ADCC) as measured in a 3H-thymidine release assay occurred after culturing the monocytes for 5-7 days. Human recombinant macrophage colony stimulating factor (CSF) (1,000 U/ml) did not increase ADCC above control levels whereas recombinant human granulocyte-macrophage colony stimulating factor, interleukin 4, and interleukin 3 were all capable of increasing ADCC. Antibodies to the CD11/CD18 integrin receptors did not significantly inhibit ADCC. When the ADCC incubation occurred in the presence of antibodies to the human Fc receptors, ADCC was inhibited significantly only by anti-FcRIII (3G8). A role for tumor necrosis factor alpha or other soluble mediators of cytotoxicity was not demonstrable in this system. These studies suggest avenues for manipulation and augmentation of macrophage-mediated antitumor ADCC.     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

Direct Delivery of a Cytotoxic Anticancer Agent into the Metastatic Lymph Node Using Nano/Microbubbles and Ultrasound

Direct injection of an anticancer agent into a metastatic lymph node (LN) has not been used as a standard treatment because evidence concerning the efficacy of local administration of a drug into a metastatic LN has not been established. Here we show that the combination of intralymphatic drug delivery with nano/microbubbles (NMBs) and ultrasound has the potential to improve the chemotherapeutic effect. We delivered cis-diamminedichloroplatinum (II) (CDDP) into breast carcinoma cells in vitro and found that apoptotic processes were involved in the antitumor action. Next, we investigated the antitumor effect of intralymphatic chemotherapy with NMBs and ultrasound in an experimental model of LN metastasis using MXH10/Mo-lpr/lpr mice exhibiting lymphadenopathy. The combination of intralymphatic chemotherapy with NMBs and ultrasound has the potential to improve the delivery of CDDP into target LNs without damage to the surrounding normal tissues. The present study indicates that intralymphatic drug delivery with NMBs and ultrasound will potentially be of great benefit in the clinical setting.     

Folate requirement for blast cell transformation in mixed leukocyte cultures.

Mixed leukocyte cultures from normal donors were set up in standard tissue culture media. Blast cell transformation was measured 6 days later by ³H-thymidine uptake and assessed qualitatively with stained smears. Media RPMI 1629 and RPMI 1640 allowed much more intense blastogenesis than Medium 199, the difference being due to the low folic acid content of Medium 199 (0.01 mg/liter) compared with RPMI 1629 (10 mg/liter) and RPMI 1640 (1 mg/liter). Further experiments indicated that folic acid increased the multiplication rate of blast cells but did not promote the induction or initial transformation phases of the mixed leukocyte reaction. The effect of folic acid on the PHA response was entirely different: ³H-thymidine uptake in 3 day PHA cultures was decreased, and there was no apparent effect on the number or percentage of blast cells seen on the smears. The reason for this difference is at present unknown, but some possibilities are discussed.     

Effect of 2'-deoxyadenosine on thymidine utilization by tradescantia pollen grains

The uptake of ³H-thymidine into pollen grains of Tradescantia paludosa was studied in the presence of 2′-deoxyadenosine. 1) Millimolar deoxyadenosine caused an immediate inhibition of incorporation of ³H-thymidine into DNA extracted with hot trichloroacetic acid. 2) The radioactivity in acid-soluble derivatives of ³H-thymidine was examined by paper chromatography and, following incubation of pollen grains in the presence of millimolar deoxyadenosine, was found to be increased several-fold in ³H-deoxythymidine triphosphate. 3) The time-course of inhibition showed that the acid-soluble derivatives of ³H-thymidine accumulated initially at a rate unaffected by deoxyadenosine, despite the nearly complete inhibition of incorporation of ³H-thymidine into DNA. This is discussed in relation to possible mechanisms of inhibition by deoxyadenosine.     

Piezo type mechanosensitive ion channel component 1 facilitates gastric cancer omentum metastasis

The peritoneum, especially the omentum, is a common site for gastric cancer (GC) metastasis. Our aim was to expound the role and mechanisms of Piezo1 on GC omentum metastasis. A series of functional assays were performed to examine cell proliferation, clone formation, apoptosis, Ca²⁺ influx, mitochondrial membrane potential (MMP) and migration after overexpression or knockdown of Piezo1. A GC peritoneal implantation and metastasis model was conducted. After infection by si-Piezo1, the number and growth of tumours were observed in abdominal cavity. Fibre and angiogenesis were tested in metastatic tumour tissues. Piezo1 had higher expression in GC tissues with omentum metastasis and metastatic lymph node tissues than in GC tissues among 110 patients. High Piezo1 expression was associated with lymph metastasis, TNM and distant metastasis. Overexpressed Piezo1 facilitated cell proliferation and suppressed cell apoptosis in GC cells. Moreover, Ca²⁺ influx was elevated after up-regulation of Piezo1. Piezo1 promoted cell migration and Calpain1/2 expression via up-regulation of HIF-1α in GC cells. In vivo, Piezo1 knockdown significantly inhibited peritoneal metastasis of GC cells and blocked EMT process and angiogenesis. Our findings suggested that Piezo1 is a key component during GC omentum metastasis, which could be related to up-regulation of HIF-1α.     

A DELAY IN THE G2 PERIOD OF CULTURED HUMAN LEUCOCYTES AFTER TREATMENT WITH RUBIDOMYCIN (DAUNOMYCIN)

Human leucocytes were cultured for 3 days at 37°C, and during this time treated with rubidomycin (also known as daunomycin) for periods up to 48 hr. The effects of this treatment were studied by examining mitotic indices, uptake of ³H-thymidine, and patterns of DNA content (measured by microdensitometry on Feulgen-stained cells). A low concentration of rubidomycin (0.1 μg/ml) caused accumulation of cells in the G₂ period, which in turn resulted in a decrease in the mitotic index. A secondary effect was a slight drop in ³H-thymidine uptake after 12 hr. Higher doses (up to 10 μg rubidomycin per ml) caused an inhibition of DNA synthesis, with accumulation of unlabelled cells between G₂ and G₂. The probable mode of action of rubidomycin, as presented by earlier authors, is the intrusion of the drug molecule between DNA strands, forming a complex with DNA, and hindering its normal folding. This is discussed with respect to the present findings.     

Time Dependent Uptake of (-)125I-Iodocyanopindolol but not (-)125I-Iodopindolol by Murine Splenic Lymphocytes

Abstract

Time dependent uptake of (-)¹²⁵I-iodocyanopindolol by intact murine splenocytes was investigated in an attempt to prevent its interference in receptor binding experiments. Four approaches were tried in an attempt to eliminate this uptake; including the use of the permeable amine chloroquine; the inclusion of catechol, chloroquine, and phentolamine into the binding assay; the addition of methylamine to the assay: and the elimination of ascorbate from the assay. None of these approaches completely eliminated the uptake of ICYP over time. Comparative studies with (-)¹²⁵I-iodopindolol showed this ligand did not have time dependent uptake and may provide for less problematic intact cell receptor assays.     

Methotrexate enhances 3T3-L1 adipocytes hypertrophy

Methotrexate (MTX) is broadly used in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA). The prevalence of metabolic syndrome (MeS) in patients with this condition is relatively high. Given the importance of adipose tissue in the development of obesity metabolic complications, this study aimed to investigate the effect of methotrexate on preadipocyte proliferation, adipogenesis, and glucose uptake by adipocytes. 3T3-L1 preadipocytes proliferation was evaluated by sulforhodamine B staining and ³H-thymidine incorporation, after 24 or 48 h of treatment with MTX (0.1 and 10 μM). Preadipocytes were induced to differentiate with an appropriate adipogenic cocktail in the presence or absence of MTX. Adipogenesis was determined by measuring lipid accumulation after staining with oil red O. ³H-Deoxyglucose (³H-DG) uptake was determined by liquid scintillation counting. MTX treatment reduced culture protein content in a concentration-dependent manner and ³H-thymidine incorporation (P?<?0.05). MTX (0.1 μM) treatment increased lipid accumulation and basal ³H-DG uptake by adipocytes (P?<?0.05). In 0.1 μM MTX-treated adipocytes, insulin stimulation did not result in an increase of ³H-DG uptake, contrarily to what was observed in control cells. These results demonstrate that methotrexate interferes with adipocyte proliferation and promotes the hypertrophic growth of adipocytes. These molecular effects may have implications on metabolic profile of RA patients treated with MTX.     

Selective inhibition of thymidine transport at low doses of the alkylating agents triethyleneiminobenzoquinone (Trenimon)

The alkylating antitumor agent triethyleneiminobenzoquinone (Trenimon) causes a rapid decrease in the incorporation of labeled thymidine into the DNA of Yoshida or Ehrlich ascites tumor cells. The effect is expressed 4 h after administration of 6 × 10⁻⁸ moles/kg of the drug to mice bearing Yoshida ascites tumors or of 6 × 10⁻⁷ moles/kg to Ehrlich ascites tumor-bearing animals, respectively. The reduced incorporation of labeled thymidine which is observed under these conditions is not due to an inhibition of DNA synthesis. DNA synthesis was measured by an isotope dilution assay after pulse-labeling with ³H-thymidine and by monitoring the increase in the total amount of DNA of the cell populations. The data demonstrate that DNA synthesis is not affected during the first 8 h after exposure to the drug. This conclusion is supported by cell kinetic measurements which indicate that the alkylating agent does not interfere with the progression of cells into the S phase, but exerts a block at the G 2 stage of the cell cycle. The reduced incorporation of thymidine into DNA is explained by a decreased transport of the nucleoside into the cells.