Effects of the garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in human colon tumour cells

Diallyl sulfide (DAS) and diallyl disulfide (DADS), major components of garlic, were used to determine inhibition of arylamine N-acetyltransferase (NAT) activity in a human colon tumour (adenocarcinoma) cell line. Two assay systems were performed, one with cellular cytosols (9000g supernatant), the other with intact bacterial cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by DAS and DADS in a dose-dependent manner in both system: that is, the greater the concentration of DAS and DADS in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax of NAT enzymes from human colon tumour cells in both systems examined. This is the first report to demonstrate that garlic components do affect human colon tumour cell NAT activity.     

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.     

Potentiation of tumour necrosis factor-mediated cell killing by VP16 on human ovarian cancer cell lines. In vitro results and clinical implications

Synergism between recombinant human tumour necrosis factor (rHuTNF) and DNA topoisomerase II inhibitor VP16 during the killing of cells has been studied in six human ovarian cancer cell lines (A2774, A2780, SW626, IGROV-1, SKOV3, Pa1) and a cervical carcinoma cell line (Me180). Studies were performed using an assay of colony formation inhibition (drug treatment for 1 h) and a growth inhibition assay (continuous exposure for 20 h). Concomitant treatment of cells with VP16+rHuTNF enhanced cell killing in all the cell lines tested--an effect observed in both short- and long-term cytotoxicity assays. This study suggests that the activity of VP16 in ovarian cancer cell lines might be enhanced by rHuTNF in in vitro models.     

Inhibitory actions of glycyrrhizic acid on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) as substrates were determined in Helicobacter pylori, collected from patients with peptic ulcers. The NAT activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. Inhibition of growth studies from H. pylori demonstrated that glycyrrhizic acid elicited dose-dependent bactericidal effect in H. pylori cultures, i.e.; the greater the concentration of glycyrrhizic acid, the greater the inhibition of growth of H. pylori. Cytosols or suspensions of H. pylori with and without selected concentrations of glycyrrhizic acid co-treatment showed different percentages of 2-AF and PABA acetylation. The data indicated that there was decreased NAT activity associated with increased glycyrrhizic acid in H. pylori cytosols and intact cells. For the cytosol and intact bacteria examinations, the apparent values of Km and Vmax were decreased after co-treated with 80 M glycyrrhizic acid. This report is the first demonstration of glycyrrhizic acid inhibition of arylamine NAT activity and glycyrrhizic acid inhibition of growth in the bacterium H. pylori.     

The tumor-selective somatostatin analog, TT2-32 induces a biphasic activation of phosphotyrosine phosphatase activity in human colon tumor cell line, SW620

Somatostatin has been demonstrated to activate phosphotyrosine phosphatases (PTPases) in pancreatic cells. In this work we studied the effect of a tumor-selective somatostatin structural derivative, TT2-32, on the PTPase activity in the SW620 human colon tumor cell line. TT2-32 caused a strong inhibition of cell proliferation. In response to TT2-32 we found a rapid and sustained increase (5-30 min) in PTPase activity showing two maxima at 0.1 and 30 microM concentrations, respectively. During short-term incubation tyrosine kinase activity was much less affected by TT2-32. TT2-32-induced activation of PTPases may be an important early step in the signaling cascade in the inhibition of cell proliferation in colon carcinomas.     

Construction of Syrian hamster lines congenic at the polymorphic acetyltransferase locus (NAT2): acetylator genotype-dependent N- and O-acetylation of arylamine carcinogens

Congenic Bio. 1.5/H-NAT2 Syrian hamster lines were constructed by introducing the NAT2r gene from MHA/SsLak inbred hamsters into a background BIO 1.5 Syrian inbred hamster line. Genetic identity of the Bio. 1.5/H-NAT2 congenic lines and nonidentity with the previously constructed Bio. 82.73/H-Pat congenic lines were determined by "DNA fingerprints" of genomic DNA derived from the different hamster lines. The N-acetylation capacity of the Bio. 1.5/H-NAT2 congenic hamster lines was clearly NAT2-dependent both in vivo and in vitro, with highest levels expressed in Bio. 1.5/H-NAT2r homozygous rapid acetylators, intermediate levels in Bio. 1.5/H-NAT2r/NAT2s heterozygous acetylators, and lowest levels in Bio. 1.5/H-NAT2s homozygous slow acetylators. The NAT2-dependent expression of N-acetyltransferase activity was evident toward p-aminobenzoic acid, 4-aminophenol, 2-aminofluorene, 4-aminobiphenyl, beta-naphthylamine, and 3,2'-dimethyl-4-amino-biphenyl in liver, kidney, colon, lung, and urinary bladder cytosols. The polymorphic acetyltransferase (NAT2) and the monomorphic acetyltransferase (NAT1) were isolated from hepatic cytosols and tested separately for their ability to catalyze arylamine N-acetyltransferase and N-hydroxyarylamine O-acetyltransferase activities. Both arylamine N-acetylation and N-hydroxyarylamine O-acetylation were clearly acetylator genotype-dependent when catalyzed by NAT2, and both were clearly acetylator genotype-independent when catalyzed by NAT1. NAT2/NAT1 activity ratios varied with the particular arylamine substrate acetylated. These studies show an important role for NAT2 acetylator genotype in Syrian hamster carcinogenic arylamine metabolism and confirm its role in the metabolic activation of N-hydroxyarylamines. The Bio. 1.5/H-NAT2 congenic lines provide a new model for investigating the precise role of the NAT2 gene locus in arylamine metabolism and toxicity.     

Individualized cardiovascular prevention: when and how far should it go?]

The antimicrobial activity of T cell-derived cytokines, especially interferon (IFN)-gamma, against intracellular pathogens, such as Chlamydia trachomatis, involves the induction of 3 major biochemical processes: tryptophan catabolism, nitric oxide (NO) induction and intracellular iron (Fe) deprivation. Since the epithelial cell is the natural target of chlamydial infection, the presence of these antimicrobial systems in the cell would suggest that they may be involved in T cell control of intracellular multiplication of Chlamydia. However, the controversy over whether these 3 antimicrobial processes are present in both mice and humans has precluded the assessment of the relative contribution of each of the 3 mechanisms to chlamydial inhibition in the same epithelial cell from either mice or humans. In the present study, we identified a Chlamydia-susceptible human epithelial cell line, RT4, that possesses the 3 antimicrobial systems, and we examined the role of nitric oxide (NO) induction, and deprivation of tryptophan or Fe in cytokine-induced inhibition of chlamydiae. It was found that the 3 antimicrobial systems contributed to cytokine-mediated inhibition of the intracellular growth of Chlamydia. NO induction accounted for approximately 20% of the growth inhibition; tryptophan catabolism contributed approximately 30%; iron deprivation was least effective; but the combination of the 3 systems accounted for greater than 60% of the inhibition observed. These results indicate that immune control of chlamydial growth in human epithelial cells may involve multiple mechanisms that include NO induction, tryptophan catabolism and Fe deprivation.     

Thiolmethyltransferase activity in the human colonic mucosa: implications for ulcerative colitis

Ulcerative colitis is associated with a selective reduction of n-butyrate oxidation by the colonic epithelial cells although the reason for this has been unclear. Colonic epithelial cell n-butyrate oxidation can be inhibited in vitro by incubation with sulphide but the role of mucosal detoxification of sulphide in the metabolic welfare of the colonic mucosa has not been examined. This study aimed to assess the role mucosal detoxification of sulphide by thiolmethyltransferase (TMT)-mediated methylation may play in protecting the healthy colonic mucosa from the adverse effects of luminal sulphide. Colonic epithelial cell suspensions from healthy human proximal (n = 9) and distal colon (n = 10) were incubated in the presence of 14C-labelled n-butyrate (5 mmol/L) alone, butyrate plus sodium hydrogen sulphide (NaHS) (1.5 mmol/L), or butyrate plus NaHS plus S-adenosyl-methionine 1,4 butane disulphonate (SAMe) (5 mmol/L). Study end points were metabolic performance (14CO2 production) and mucosal TMT activity. Incubation with NaHS induced a significant inhibition of 14CO2 production compared with control incubations (P < 0.001) which was similar for proximal and distal colonic cell suspensions. S-adenosyl-methionine 1,4 butane disulphonate reversed this effect completely in proximal but not in distal cell incubations, suggesting a greater susceptibility of the distal colon to the sulphide effect. Although median whole mucosal TMT values did not differ between proximal and distal colonic mucosa, a non-normal distribution of distal TMT values was observed. However, neither the degree of sulphide inhibition of control 14CO2 production nor the degree to which SAMe reversed this inhibition correlated with whole mucosal TMT activity. The study concluded that regional variation exists in TMT activity in the human colon but whilst methylation appears to protect colonic epithelial cells against sulphide-induced inhibition of n-butyrate oxidation, this cannot be directly correlated with mucosal TMT activity.     

Effects of garlic compounds diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Arylamine N-acctyltransferase (NAT) activities with p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients. Two assay systems were performed, one with cellular cytosols, the other with intact cell suspensions. Cytosols or suspensions of H. pylori with or without specific concentrations of diallyl sulfide (DAS) or diallyl disulfide (DADS) co-treatment showed different percentages of 2-AF and PABA acetylation. The data indicated that there was decreased NAT activity associated with increased levels of DAS or DADS in H. pylori cytosols and suspensions. Viability studies on H. pylori demonstrated that DAS or DADS elicited dose-dependent bactericide affects on H. pylori cultures. The data also indicated that DAS and DADS decreased the apparent values of K(m) and Vmax of NAT enzyme from H. pylori in both systems examined. This report is the first demonstration that garlic components can affect H. pylori growth and NAT activity.     

Longitudinal distribution of arylamine N-acetyltransferases in the intestine of the hamster, mouse, and rat. Evidence for multiplicity of N-acetyltransferases in the intestine

Experimental and clinical evidence indicates that AcCoA:arylamine N-acetyltransferases (NATs; EC 2.3.1.5) are involved in the bioactivation and inactivation of a wide variety of arylamine, hydrazine, and carcinogenic arylamine xenobiotics. Longitudinal distribution of NATs in the intestine of the hamster, mouse, and two strains of rat was examined utilizing the model arylamine substrates procainamide(PA) and p-aminobenzoic acid (PABA) for the monomorphic (NAT1) and polymorphic (NAT2) enzymes in the rodent. NAT1 and NAT2 were distributed quite differently in each species examined. In particular, rat intestinal NATs were distributed equally throughout the intestinal tract. In contrast, hamster intestinal NATs decreased in activity from the proximal small intestine to the distal large intestine. Mouse NAT2 activity was highest in the cecum, whereas NAT1 was highest in the proximal small intestine. Although these model substrates have been shown to be selective for NATs, they are not specific. Therefore, a series of biochemical studies were undertaken to evaluate NAT multiplicity in the intestine of the F-344 rat. To assess multiplicity of NAT expression, selective inhibition, differential sensitivity to heat inactivation, and kinetic analysis were performed on intestinal cytosol. Eadie-Hofstee transformation of PA N-acetylation yielded a curvilinear plot indicative that a low affinity-high capacity enzyme aside from NAT1 (presumably NAT2) was contributing to PA N-acetylation activity. PA activity was found to exhibit approximately 4- to 5-fold greater thermostability than PABA activity. Furthermore, PA acetylation could be inhibited selectively with vinyl fluorenyl ketone (2.5 to 5 microM) but not with methotrexate (up to 2 mM). Taken together, these studies suggest the expression of both NAT1 and NAT2 in the intestine of the F-344 rat.     

Inhibition of tolbutamide 4-methylhydroxylation by a series of non-steroidal anti-inflammatory drugs in V79-NH cells expressing human cytochrome P4502C10

1. To study the role of cytochrome P4502C10 in the metabolism of the non-steroidal antiinflammatory drugs (NSAIDs) diclofenac, phenylbutazone, fenoprofen, ibuprofen, flurbiprofen, ketoprofen and naproxen, a cell line was developed stably expressing CYP2C10 cDNA. A retroviral vector construct, containing a human CYP2C10 cDNA, was transfected in V79-NH Chinese hamster lung cells by calcium phosphate co-precipitation. Sublines stably expressing human cytochrome P450 cDNA were established by selection with the neomycin analogue G418. 2. Enzymatic activity of CYP2C10 was detected by 4-methylhydroxylation of tolbutamide. This activity was inhibited to background levels by preincubation with the CYP2C9/10 inhibitor sulphaphenazole. 3. Preincubations with the NSAIDs ketoprofen, phenylbutazone, flurbiprofen and diclofenac (all 250 microM) caused a decrease in 4-methylhydroxylation of tolbutamide (500 microM), significantly different from control values (p < 0.05). Inhibition of this activity was not seen in preincubations with the NSAIDs fenoprofen, ibuprofen and naproxen (250 microM). 4. The V79-NH CYP2C10 cell line we have developed has been shown to be a useful tool to predict drug-drug interactions.     

Successful islet autotransplantation in humans: functional insulin secretory reserve as an estimate of surviving islet cell mass

Bafilomycin A1, a specific inhibitor of vacuolar type H(+)-ATPase, can inhibit the growth of a variety of cultured cells in a dose-dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitro and in vivo was examined using an MTT assay and an in vivo tumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan-1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan-1 cell line tumour received 4 weeks of bafilomycin A1 (1.0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P < 0.05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan-1 human pancreatic cancer cells through apoptosis.     

Determinants of trimetrexate lethality in human colon cancer cells

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.     

A new screening system for NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent

NAD(P)H:quinone oxidoreductase (NQO1; DT-diaphorase) is elevated in certain tumors, such as non-small cell lung cancer (NSCLC). Compounds such as mitomycin C and streptonigrin are efficiently bioactivated by NQO1 and have been used in an enzyme-directed approach to chemotherapy. Previously, 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ) was identified as a potential antitumor agent based on its high rate of bioactivation by human NQO1 and its selective cytotoxicity to cells containing elevated NQO1. RH1, a water-soluble analogue of MeDZQ synthesized in this work, was a better substrate for recombinant human NQO1 than the parent compound. RH1 was, correspondingly, more cytotoxic to human tumor cells expressing elevated NQO1 activity (H460 NSCLC and HT29 human colon carcinoma), as measured by 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium assay, than it was to cells deficient in NQO1 activity (H596 NSCLC and BE human colon carcinoma). RH1 exhibited a greater selective toxicity (ratio of IC50s in H596:H460 and BE:HT29) to cells with elevated NQO1 activity relative to MeDZQ. Additionally, we report the establishment of a stable line of BE human colon carcinoma cells transfected with wild-type human NQO1 (BE-NQ7). BE cells are devoid of NQO1 activity due to a homozygous point mutation in the NQO1 gene. In comparison to the parental cell line, RH1, MeDZQ, and mitomycin C were significantly more cytotoxic to BE-NQ7 cells (17-, 7-, and 3-fold, respectively), confirming that the presence of NQO1 is sufficient to increase cytotoxicity of these antitumor quinones. These data suggest that RH1 may be an effective NQO1-directed antitumor agent for the therapy of tumors with elevated NQO1 activity, such as NSCLC.     

1H MRS markers of tumour growth in intrasplenic tumours and liver metastasis induced by injection of HT-29 cells in nude mice spleen

We have characterized, by in vitro magnetic resonance spectroscopy (MRS), the metabolite pattern of perchloric acid (PCA) extracts of intrasplenic tumours and hepatic metastasis, produced by intra-spleen injection of the human colorectal carcinoma cell line HT-29 and its metastatic variant HT-29 MMM into nude mice. Our aim was to gain further understanding of colorectal tumour metabolism as a basis for future in vivo studies of human colon cancer by 1H MRS. Metabolite PCA extract analysis showed a good reproduction of the spectral pattern observed in human primary colon tumours, while they were very different from the spectral pattern of the host tissues (spleen and liver). The main differences between host and tumour tissues involved taurine, phosphocholine (PC), phosphoethanolamine (PE), creatine, glycogen and glucose. Creatine is the most promising marker to follow tumour growth because of its practical absence in the nude mice host tissues. Detection of variable levels of this compound and of taurine in hepatic foci in man, are suggested as possible diagnostic markers. No correlation could be found between spectral pattern differences and the different ability to metastasize of the two HT-29 cell lines used. Furthermore, indirect evidence for a functional link between taurine and myo-inositol in colon tumour cells is presented. In summary, our data suggest that the nude mice model may be a suitable system for the MRS study of the changes taking place in host tissues upon tumour progression.     

Thrombin enhances the adhesion and migration of human colon adenocarcinoma cells via increased beta 3-integrin expression on the tumour cell surface and their inhibition by the snake venom peptide, rhodostomin

The interactions between tumour cells and the microvasculature, including the adhesion of tumour cells to endothelium and extracellular matrix (ECM) as well as their migratory ability, are prerequisites for metastasis to occur. In this study we showed that thrombin is capable of enhancing in vitro tumour cell metastatic potential in terms of adhesive properties and migratory response. Following exposure to subclotting concentrations of thrombin, SW-480 human colon adenocarcinoma cells exhibited increased adhesion to both the endothelium and ECM component (i.e. fibronectin). Likewise, the pretreatment of thrombin enhanced the migratory ability of SW-480 cells. The enhanced adhesion was significantly inhibited by complexing of thrombin with its inhibitor hirudin, or by serine proteinase inhibition with 3,4-DCI, but was unaffected by pretreatment of tumour cells with actinomycin D or cycloheximide. The effect of thrombin resulted in an upregulated cell-surface expression of beta 3 integrins, a group of receptors mediating interactions between tumour cells and endothelial cells, and between tumour cells and ECM. Antibodies against beta 3 integrins effectively blocked both the enhanced adhesion and migration. This thrombin-mediated up-regulation of beta 3 integrins involved the activation of protein kinase C (PKC) as thrombin-enhanced adhesion was diminished by PKC inhibition. Rhodostomin, an Arg-Gly-Asp-containing antiplatelet snake venom peptide that antagonises the binding of ECM toward beta 3 integrins on SW-480 cells, was about 600 and 500 times, more potent that RGDS in inhibiting thrombin-enhanced adhesion and migration respectively. Our data suggest that PKC inhibitors as well as rhodostomin may serve as inhibitory agents in the prevention of thrombin-enhanced metastasis.     

Differential effect of acetyltransferase expression on the genotoxicity of heterocyclic amines in CHO cells

We earlier developed the Chinese hamster ovary UV5P3 cell line that expresses cytochrome P4501A2 and lacks nucleotide excision repair for studying metabolism and mutagenicity of heterocyclic amines. The Chinese hamster ovary UV5P3 cells are approximately 50-fold more sensitive to the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) than 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), another genotoxic compound found in cooked food, with respect to cytotoxicity and mutation induction at the adenine phosphoribosyltransferase (aprt) locus. To test the hypothesis that the important missing activity in our CHO system for IQ genotoxicity was acetyltransferase, we transfected the UV5P3 cells with cDNA plasmids of either the human NAT2 N-acetyltransferase gene or a bacterial O-acetyltransferase gene. Functionally transformed clones were determined by the differential cytotoxicity assay using IQ, and confirmed by measuring the enzyme activity with isoniazid as substrate. Two clones designated 5P3NAT2 and 5P3YG (expressing human and bacterial transferases, respectively) were characterized. Both cell lines were sensitive to killing by IQ at concentrations as low as 4 ng/ml. Based on the D37 value, the dose that reduced the survival to 37% relative to untreated controls, the acetyltransferase expressing lines showed approximately 1000-fold increase in sensitivity to the killing effect of IQ over the parental UV5P3 cell line. The same dramatic change in sensitivity was also seen in mutation response at the aprt locus and with chromosomal aberrations and sister chromatid exchanges. In contrast, these cell lines showed cytotoxicity to PhIP similar to that of the parental line UV5P3. These results suggest that PhIP does not require acetyltransferase for metabolic activation leading to genotoxicity in these cells. These new cell lines constitute a sensitive cell system for assessing genotoxicity of compounds requiring metabolic activation by both P450IA2 and acetyltransferase, as well as for studying the molecular processes by which DNA damage can lead to mutation and cancer.     

The in-vitro generation of dendritic cells from blast cells in acute leukaemia

Recent clinical observations indicate that ibuprofen may alleviate the radiation-induced dysuria that almost invariably occurs during radiation therapy for prostate cancer. Because the use of ibuprofen could consequently become common during radiation therapy for prostate cancer, we have been interested in the potential interactions between ibuprofen and ionizing radiation on prostate tumor cells. The effects of gamma-irradiation and/or ibuprofen on PC3 and DU-145 human prostate carcinoma cells were evaluated in vitro using three model systems. Clonogenic survival was determined by plating cells 24 h after treatment of nearly confluent monolayers. Analysis of cell growth, cell detachment, and apoptotic cell death was carried out over a period of up to 9 days after treatment of PC3 and DU-145 monolayers. The effect of ibuprofen and/or radiation was also probed by observing the inhibition of growth of established PC3 and DU-145 colonies that were treated on the 14th day of colony growth. Ibuprofen enhanced the radiation response of prostate cancer cells in all three in vitro models. Both the cytotoxic and radiosensitizing effects of ibuprofen seem to require concentrations that are higher than those reported to inhibit prostaglandin synthesis, suggesting that other molecular mechanisms may be responsible for ibuprofen cytotoxicity.     

Taxol-mediated augmentation of CD95 ligand-induced apoptosis of human malignant glioma cells: association with bcl-2 phosphorylation but neither activation of p53 nor G2/M cell cycle arrest

The anti-tumour alkaloid taxol shows strong cytotoxic and antiproliferative activity in two human malignant glioma cell lines, T98G and LN-229. CD95 (Fas/APO-1) ligand is a novel cytotoxic cytokine of the tumour necrosis factor (TNF) family that exerts prominent antiglioma activity. At clinically relevant taxol concentrations of 5-100 nM, taxol and CD95 ligand showed significant synergistic cytotoxicity and growth inhibition. High concentrations of taxol induced G/M cell cycle arrest in both cell lines. The synergy of taxol and CD95 ligand was independent of cell cycle effects of taxol as synergy was achieved at much lower taxol concentrations than G2/M arrest and as cell cycle effects of taxol were unaffected by co-exposure to CD95 ligand. Similarly, high concentrations of taxol were required to induce p53 activity in the p53 wild-type cell line LN-229. This effect was not modulated by CD95 ligand, suggesting that synergy is also independent of p53 activation. However, taxol induced a mobility shift of the bcl-2 protein on immunoblot analysis, indicative of bcl-2 phosphorylation. Bcl-2 phosphorylation on serine was confirmed by immunoprecipitation and phosphoserine immunoblot analysis. Considering (1) that phosphorylation of bcl-2 interferes with its heterodimerization with bax and (2) the inhibition of CD95-mediated apoptosis by bcl-2, we propose that taxol sensitizes malignant glioma cells to CD95 ligand by increasing the functional bax/bcl-2 rheostat in favour of bax and thus cell death.     

Pre-clinical evaluation of a novel chloroethylating agent, Clomesone

The in vitro activity of the novel chloroethylating agent, Clomesone, was investigated in a panel of established murine and human tumour cell lines. In vivo anti-tumour activity was examined against three transplantable adenocarcinomas of the mouse colon and in vivo bone marrow toxicity was assessed using a spleen colony forming unit assay. The pharmacokinetic behaviour of the drug in vivo and drug stability in vitro was analysed by gas chromatography with electron capture detection. Clomesone exhibited no activity in vitro against the majority of cell lines derived from solid human colorectal carcinomas. Anti-tumour activity against the murine tumours in vivo was not impressive and was accompanied by myelosuppression. Pharmacokinetic data suggested that the lack of in vivo activity was due to the failure to achieve effective anti-neoplastic drug concentrations at the tumour site. It was concluded that this study found no evidence to suggest that Clomesone was toxicologically more selective than the chloroethylnitrosoureas.