Inhibition Of Colony Growth Research Articles

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.

Involvement of Fas-Mediated Apoptosis in the Inhibitory Effects of Interferon-α in Chronic Myelogenous Leukemia

AbstractInterferon-α (IFN-α) is an established treatment for chronic myelogenous leukemia (CML) in chronic phase, but the mechanism of its antileukemic activity is not clear. One possible mechanism of action might include the induction of apoptosis, and especially Fas-mediated cell killing may play an important role in the elimination of malignant cells. We investigated Fas receptor (Fas-R) expression and the consequences of Fas-R triggering in CML patients. Using two-color flow cytometry, we found a significantly higher number of Fas-R–expressing CD34+ cells in the bone marrow (BM) of CML patients compared with normal subjects. We have previously shown that IFN-γ induces Fas-R expression on CD34+ cells; in this study, we investigated whether IFN-α induces Fas-R expression on CML progenitor cells. Dose-dependent induction of Fas-R expression was observed after IFN-α stimulation of CD34+ cells from CML BM. In methylcellulose culture, IFN-α alone at a therapeutic concentration showed only marginal antiproliferative effects on both normal and CML BM progenitors. In contrast, a Fas-R agonist, the anti-CD95 monoclonal antibody CH11, inhibited colony formation from normal progenitors, and the inhibition was even stronger on CML progenitors. When CML BM cells were cultured in the presence of IFN-α, Fas-R–mediated inhibition of colony growth was potentiated in a dose-dependent fashion, consistent with IFN-α induction of Fas-R expression. This functional effect did not require the presence of accessory cells, since similar results were obtained with purified CD34+ cells. In suspension cultures, we demonstrated that suppression of CML hematopoiesis by IFN-α and Fas-R agonist was exerted through Fas-R–mediated induction of apoptosis. Our findings suggest that the Fas-R/Fas-ligand system might be involved in the immunologic regulation of CML progenitor growth and that its effect can be amplified by IFN-α.

Sensitivity of Mycosphaerella fijiensis, Causal Agent of Black Sigatoka of Banana, to Propiconazole

ABSTRACT One hundred monoascosporic isolates of Mycosphaerella fijiensis were collected in February and November 1994 from each of two banana (Musa spp.) plantations in Costa Rica. Locations at San Pablo and Coopecariari had been sprayed with propiconazole for the past 7 years to control black Sigatoka. One hundred monoascosporic isolates from a third location, San Carlos, with no history of fungicide use, also were tested for sensitivity to propiconazole. Fifty percent effective concentration (EC(50)) values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. In the February sample, the mean EC(50) values for San Pablo and Coopecariari populations were 0.06 and 0.05 mug a.i. ml(-1), respectively, which were not statistically different (P = 0.05). The mean EC(50) value of the population at San Carlos was 0.008 mug a.i. ml(-1), which was significantly lower (P = 0.001) than the mean EC(50) values obtained at San Pablo and Coopecariari. Frequency distributions of EC(50) values of isolates from the three populations collected in February showed that 80% of isolates from San Pablo and Coopecariari had EC(50) values greater than the highest EC(50) value from San Carlos, indicating a significant shift in reduced sensitivity to propiconazole. Isolates collected in November 1994, after eight treatments of propiconazole at San Pablo and Coopecariari, showed a significant increase in mean EC(50) values compared with the means observed in February. The high proportion of isolates with reduced sensitivity to propiconazole may account for the unsatisfactory control of black Sigatoka between 1992 and 1993 in the two banana plantations at San Pablo and Coopecariari.

Cytotoxic Action of Carboxyborane Heterocyclic Amine Adducts

The heterocyclic carboxyborane amines were found to be potent cytotoxic agents in the murine L1210 lymphoid leukemia and human HeLa suspended carcinoma cells. These agents were observed to inhibit HeLa DNA topoisomerase II activity ~ 200 μM and L1210 topoisomerase II activity ≥ 100 μM. These agents did not cause DNA protein linked breaks themselves, but upon incubation for 14-24 hr did enhance the ability of VP-16 to cause cleavable complexes. The heterocyclic amineboranes inhibited DNA synthesis and caused DNA strand scission. They were additive with VP-16 in affording these results as well as inhibiting colony growth of L1210 cells after co-incubation for 1 hr. The agents inhibited in vitro PKC phosphorylation of both L1210 lymphoid leukemia and human topoisomerase II enzyme.

Murine marrow stromal response to myelotoxic agents in vitro.

Previous studies have shown that the haemopoietically active murine long-term bone marrow culture (LTBMC) is a useful model for studying drug-induced suppression and recovery of myelopoiesis. We studied the effects on stromal morphology and stromal progenitors, assessed as colony forming unit-fibroblasts (CFU-F), of the addition of either the antimetabolite methotrexate (MTX) or the betalactam ceftazidime (CEF) to LTBMC. The examination of 500 micrograms/ml CEF-treated cultures revealed a stroma that appeared empty, with modest reduction in total stromal counts, and significant decreases in fat-containing and endothelial cells. In contrast, treatment with 1 microM MTX for 1 week initially caused minimal morphologic stromal changes, thereafter total stromal cell count as well as fibroblastoid, endothelial, fat containing and macrophage cells significantly increased. Haemopoiesis and the stroma recovered. Both CEF and MTX reversibly suppressed stromal progenitor cells in LTBMC. When added directly to CFU-F cultures, the concentrations resulting in a 50% colony growth inhibition were 214 micrograms/ml for CEF and 375 nM for MTX. These results suggest that CEF, but not MTX, has direct toxic effects on the stroma of established LTBMC. Stromal cell increase following MTX treatment probably indicates a stromal response that may contribute to haemopoietic cell recovery.

Tests of fungicides for post-germination activity against Nectria galligena, causal agent of canker and fruit rot of apple

Twelve fungicides were screened in vitro for post-germination activity against Nectria galligena, the causal agent of European apple canker and Nectria fruit rot. Colony size was recorded 5 and 12 days after the application of fungicides. Fungicides differed in their ability to inhibit colony growth. Type of fungicide and concentration interacted. In general, inhibition was greater the higher the concentration of fungicide. Also, the shorter the delay (0, 6 and 30 h) between seeding the plates with conidia and applying the fungicides, the greater the inhibition, although this effect was far less than that due to concentration. Four fungicides were selected for further in vitro testing at lower concentrations and longer delays (36 and 96 h) after seeding plates with conidia. On the basis of these in vitro screens, carbendazim, fenpropimorph and prochloraz, each with and without a chemical penetrant, were applied to pruned shoots as curative treatments on potted apple trees in a polythene tunnel. These fungicides failed to prevent infection when applied 48 h after the inoculation of fresh pruning cuts. In a second experiment, these fungicides, especially prochloraz, slightly reduced the incidence of cankered shoots and lengthened the incubation period when applied 36 h after inoculation of 1-day-old pruning cuts. The results are discussed in relation to the rate of the wound-healing process, which is likely to be a factor influencing the degree of contact between fungicides and the invading fungus.

Tirapazamine (SR 4233) interrupts cell cycle progression and induces apoptosis.

Tirapazamine (Tira), a bioreductive agent, is highly toxic to cells under low oxygen conditions. Since active investigations of this agent are focusing on its potential as an adjunct of radiotherapy to improve overall effects on radioresistant hypoxic tumor cells, understanding its toxic mechanisms under aerobic conditions is important to the clinical application of this agent. Tira-treated V79 Chinese hamster cells were tested for cytotoxicity by colony assay and growth inhibition by the MTT assay. The survival of V79 cells after being exposed to 100 microM of Tira for 2 h was about 78% of untreated controls. The mitotic cell counts of V79 cells approached zero after 4 h treatment of Tira at 100 microM or 3 h at 300 microM. The fragmentation pattern of DNA isolated from cells 2 h after 300 microM Tira treatment showed characteristics of apoptotic cells. The induction of apoptosis by Tira was also detected by flow cytometric analysis and microscopic observation. These effects of Tira may be part of underlying toxic mechanisms to cells (including normal cells) under aerobic conditions.

Fibronectin Fibrils and Growth Factors Stimulate Anchorage-Independent Growth of a Murine Mammary Carcinoma

Stromal cells are important regulators of mammary carcinoma growth and metastasis. We have previously shown that a 3T3-L1 adipocyte cell line secretes hepatocyte growth factor (HGF), which stimulates proliferation of a murine mammary carcinoma (SP1) in monolayer cultures (DNA Cell Biol.13, 1189–1897, 1994). We now examine the role of growth factors and the extracellular matrix protein fibronectin in stimulation of anchorage-independent growth of SP1 cells. Purified transforming growth factor-β (TGF-β) stimulated significant colony growth in soft agar cultures, whereas HGF had a lesser effect. Analysis by confocal microscopy revealed that carcinoma cell colonies contained extracellular microfibrils composed of fibronectin. Partial depletion of fibronectin from 7% FBS/agar cultures reduced the number of colonies; colony growth could be recovered by adding back exogenous fibronectin. Addition of the 70-kDa N-terminal fragment of fibronectin, which inhibits fibronectin fibril formation, reduced growth of SP1 cell colonies, but an 85-kDa fragment containing the cell binding domain did not inhibit colony growth. These findings indicate that deposition of extracellular fibronectin fibrils is necessary, but not sufficient, for anchorage-independent growth of SP1 mammary carcinoma cells; growth factors are also required. SP1 cells had less fibronectin mRNA and secreted less fibronectin protein under anchorage-independent conditions than under anchorage-dependent conditions, as determined by Northern blotting and immunoprecipitation analysis. Thus, both growth factors (HGF and TGF-β) and fibronectin may be important regulators of paracrine stimulation by stromal cells of anchorage-independent growth of mammary carcinoma cells.

Hydroxy-anthraquinones as antimalarial agents

A series of hydroxy- and polyhydroxy-anthraquinones were screened for inhibitory activity against the malarial parasite, Plasmodium falciparum. Rufigallol demonstrated the most potent effects with a 50% inhibitory concentration (IC 50 value of ∼10.5 ng/ml (∼35 nM). Deleterious effects were exerted by rufigallol toward bone marrow progenitor cells at concentrations≥ 10 μM (3 μg/ml) where ∼30% suppression of colony growth was noted. Taking into consideration its potency and relative lack of toxicity, we believe that rufigallol should be advanced for in vivo studies. At the very least, rufigallol represents a simple, inexpensive lead drug for the development of more potent analogs.

The biodegradation of poly-3-hydroxyalkanoates, PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively ‘long’ alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.

Interferon‐γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development

Interferon-gamma (INF-gamma) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INF gamma required to suppress murine burst forming unit-erythroid (BFU-E) growth was significantly less than that required to suppress colony forming unit-erythroid (CFU-E) growth. INF gamma acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU-E culture initiation. Inhibition was progressively less if INF gamma addition was delayed after culture initiation. The effects of INF gamma on BFU-E did not require the presence of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF alpha), or granulocyte macrophage colony stimulating factor (GM-CSF), as its effects were not neutralized by monoclonal antibodies against IL-1 alpha, TNF alpha, or GM-CSF. This applied whether INF gamma was added to culture with individual antibodies or with a combination of all three antibodies. INF gamma was not required for IL-1 alpha- or TNF alpha-induced suppression of BFU-E, as their effects were not neutralized by a monoclonal anti-INF gamma antibody. In contrast, GM-CSF-induced suppression of BFU-E was negated by the simultaneous addition of anti-INF gamma. We have previously shown that the addition of TNF alpha does not suppress BFU-E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INF gamma that does not inhibit and increasing concentration of TNF alpha were added to culture, suggesting a synergistic effect between INF-gamma and TNF alpha. These observations suggest that INF gamma is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INF gamma is also involved in GM-CSF-induced inhibition of BFU-E colony growth.

Modulation of growth and differentiation of eosinophils from human peripheral blood CD34 + cells by IL5 and other growth factors

Small numbers of CD34 + primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34 + cells. CD34 + cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34 + cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3 + GM-CSF + IL5), the addition of interferon-α or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-γ, tumor necrosis factor-α, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34 + cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10 4 cells plated at Day 3 and 134 colonies/10 4 cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5 independent. Using reverse-transcriptase polymerase chain reaction, the mRNA encoding the eosinophil-specific protein eosinophil peroxidase (EPO) was not detected in Day 0 CD34 + cells, but was demonstrated by Day 3 of culture. We conclude that within 3 days of culture, peripheral blood CD34 + cells can become committed to the eosinophil lineage as demonstrated by responsiveness to IL5 and production of EPO transcripts.

Hydroxyurea enhances 3′‐azido‐3′‐deoxythymidine (AZT) cytotoxicity in human chronic myeloid leukemia models*

In this report we have evaluated the cytotoxic activity of 3'-azido-3'-deoxythymidine (AZT) used in combination with hydroxyurea (HU), an agent which disrupts de novo thymidylate synthesis. In 2 chronic myeloid leukemia (CML) cell lines, K562 and RWLeu4, the IC50 of AZT was 8 mumol/l and 28 mumol/l respectively, after a 5-day exposure, and the IC50 of HU was 80 mumol/l and 70 mumol/l respectively. In the presence of various concentrations of HU (1 mumol/l-100 mumol/l) the IC50 of AZT in both cell lines was significantly reduced and subsequent isobologram analysis revealed synergistic activity. Similarly, analysis of [3H]AZT incorporation into the DNA fraction of these cells indicated that exposure to AZT+HU resulted in an increased incorporation of AZT into DNA when compared to incubation in AZT alone. Biochemically, this effect appeared to be related to a decrease in dTTP pools caused by HU. The combination AZT+HU has also been demonstrated to exert a synergistic effect in inhibiting colony growth of bone marrow granulocyte-macrophage progenitors (CFU-GM) from patients affected by Ph1+ CML in chronic phase. These results are promising in view of a possible in vivo utilization of this drug combination.

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line.

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF beta-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U-343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

Combined antileukemic activity of pIXY 321 and Ara-C against human acute myeloid leukemia cells.

Prolonged administration of conventional (100 mg/m2/day) or low dose Ara-C (20 mg/m2/day) has been associated with significant clinical antileukemic effects in AML and myelodysplastic syndromes. These doses and schedules of Ara-C yield plasma Ara-C concentrations in the range of 10 to 100 nM. Utilizing concentrations and a schedule of Ara-C treatment, representative of Ara-C exposures in these clinical situations, we performed in vitro studies to examine the effects of co-treatment with pIXY 321 on Ara-C induced apoptosis and Ara-C-mediated colony growth inhibition of human myeloid leukemia HL-60 cells. Significantly greater internucleosomal DNA fragmentation, higher percentage of morphologically recognizable apoptotic cells and increased colony growth inhibition were observed following treatment with 100 versus 10 nM Ara-C for 5 days. Simultaneous exposure to 10 ng/ml pIXY 321 resulted in significantly increased colony growth inhibition as well as DNA fragmentation and apoptosis due to 10 nM but not 100 nM Ara-C. These concentrations of Ara-C inhibited c-myc and did not induce c-jun mRNA expression. These effects of Ara-C on c-myc and c-jun expressions were not influenced by co-treatment with pIXY 321. Neither treatment with pIXY 321 or Ara-C alone, nor co-treatment with pIXY 321 and Ara-C, significantly altered the intracellular p26BCL-2 levels in HL-60 cells. These results indicate that co-treatment with pIXY 321 significantly increases low dose Ara-C-induced apoptosis and thereby its antileukemic activity.

High-level heterologous gene expression in Saccharomyces cerevisiae from a stable 2μm plasmid system

The best candidate for a high-copy-number and mitotic stability expression system in yeast is the endogenous 2μm plasmid. Nevertheless, derivatives of the 2μm plasmid typically exhibit lower copy numbers and require selection for adequate maintenance within cells. We report the construction and utilization of an efficient heterologous gene expression system containing a 4.5-kb inducible expression cassette inserted into the 2μm plasmid and selected in cells utilizing a carrier plasmid which is subsequently lost via FRT/Flp recombination. The non-selectable 2μm plasmid, containing the cassette, was found to be stably maintained in cells, without selection, at high copy number. The dynamics of resolution and partitioning of this plasmid were analyzed during the course of 50 generations of growth under non-selective conditions. The heterologous lacZ reporter gene coding for β-galactosidase (βGal) is driven by the hybrid, galactoseinducible promoter GAL10::pMFα 1. Upon induction, βGal was secreted into the periplasm and culture supernatant at levels which could be detected directly from Coomassie blue-stained SDS-PAGE. Furthermore, plasmid-containing cells could be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal solid medium. The cassette was designed for direct, high-level, inducible expression of cloned genes downstream from the MFα1 signal sequence, with or without a C-terminal lacZ fusion. This vector represents the first demonstration of a non-selectable, mitotically stable, episomal plasmid system capable of expressing recombinant proteins at high levels. By supplanting the need for synthetic medium, this system could provide both an efficient and cost-effective means of generating recombinant protein at either the laboratory or large-scale level.

Direct and indirect effects of human interferon alpha on renal cell carcinoma: a new in vitro assay system for evaluating cytokine-mediated antitumor effects.

A highly purified natural alpha-interferon (nIFN alpha) was tested in vitro for direct and indirect antiproliferative activity against renal cell carcinoma (RCC), using a modified human tumor clonogenic assay and clinically achievable concentrations. In preclinical experiments, the indirect (cytokine-mediated) antiproliferative activity of nIFN alpha was investigated using ACHN cells (established human RCC cell line). Continuous exposure to nIFN alpha at concentrations of more than 5 IU/ml in the presence of feeder cells (a mixture of 5 x 10(4) monocytes/dish and 5 x 10(5) lymphocytes/dish, obtained from healthy donors) significantly inhibited colony formation of ACHN cells in comparison with growth inhibition in the absence of feeder cells (P < 0.05). Various cytokines were measured in the supernatants lying over the medium on the feeder-layer agarose containing the same conditioned feeder cells. With IFN alpha at 500 IU/ml, tumor necrosis factor alpha (TNF alpha) and IFN gamma were detected at markedly high levels for 2-24 h. Neutralizing anti-TNF alpha monoclonal antibody significantly reduced the indirect antiproliferative activity. Using our modified human tumor clonogenic assay technique, sufficient numbers of colonies for drug testing were observed in 19 of 31 surgical specimens (61.3%). In these clinical materials, nIFN alpha at a clinically achievable concentration (50 IU/ml) significantly inhibited colony growth in the presence of feeder cells consisting of 5 x 10(4) monocytes/dish and 5 x 10(5) lymphocytes/dish, obtained from the patient whose tumor was examined (P < 0.05). In colony-forming cases, a significant correlation between the percentage colony survival and TNF alpha concentration in the supernatant was observed (r = -0.95, P < 0.01). These results suggest that this assay system may be an appropriate technique for evaluating the antiproliferative activities of nIFN alpha involving cytokine-mediated action, and that TNF alpha may play an important role in this cytokine-mediated activity.

Inhibition of head and neck tumor cell colony growth by lymphokine activated killer cells.

The antiproliferative effect of lymphokine activated killer (LAK) cells on head and neck tumor cells has not previously been elucidated. We studied the inhibitory effects of recombinant interleukin-2 activated lymphocytes on tumor colony formation in semisolid agar, using head and neck tumor cells prepared from established tumor cell lines (K562, HT29, HLaC78) and xenografted head and neck squamous cell carcinomas on nude mice (XKN, XLL, XFL, XKF). LAK cells demonstrated a significant inhibitory effect on colony formation. The effects of LAK cells on cultured tumor cell lines were evaluated in a dose dependent manner using effector: target ratios. In addition, the colony formation of tumor cells derived from xenografted nude mouse was inhibited by LAK cells. These results suggest that LAK cells generate an antiproliferative effect on head and neck tumor cells.

Effect of Hemopoietic Growth Factors G-CSF and pIXY 321 on the Activity of High Dose ARA-C in Human Myeloid Leukemia Cells

Recently, high dose Ara-C (HIDAC) has been shown to induce leukemic cell death in vitro by the alternative process of programmed cell death (PCD) or apoptosis which correlates with the inhibition of their clonogenic survival. Since co-treatment with hemopoietic growth facts (HGFs) GM-CSF and IL-3 have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells, we examined the effect of HGFs pIXY 321 (a GM-CSF/IL3 fusion protein) and G-CSF on HIDAC induced PCD and related gene expressions as well as HIDAC mediated colony growth inhibition of human myeloid leukemia cells. Treatment with G-CSF or pIXY 321 alone for up to 24 hours neither suppressed nor induced PCD in HL-60 or KG-1 cells. However, exposure to either of the HGFs for 20 hours followed by a combined treatment for 4 hours with HIDAC plus either of the HGFs versus HIDAC alone significantly enhanced the intracellular Ara-CTP accumulation and the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of C-jun expression but a significant repression in BCL-2 and c-myc expressions. In addition, the treatment with either of the HGFs plus HIDAC versus HIDAC alone produced a significantly greater inhibition of the clonogenic survival of the myeloid leukemia cells. These findings underscore an additional mechanism of leukemic cell death induced by HIDAC which can be modulated by the HGFs to improve the antileukemic activity of HIDAC.

Granulocyte-Macrophage Colony-Stimulating Factor/Interleukin-3 Fusion Protein (pIXY 321) Enhances High-Dose Ara-C–Induced Programmed Cell Death or Apoptosis in Human Myeloid Leukemia Cells

AbstractHigh dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.