Biomedical subjects
M D'Incalci
Publications and source records attributed to M D'Incalci.
O6-methylguanine inhibits the binding of transcription factors to DNA.
To study the effect of methylation of O6-guanine on the binding of cellular factors to different DNA sequences, modified oligonucleotides were constructed, in which O6-Methylguanine (O6-MeG) replaced some guanines. The DNA sequences utilized were: the region of the c-fos promoter containing the binding site for serum response factor (SRF); the region of the HIV LTR containing two binding sites for the transcription factor NF kappa B; the region of the HIV LTR containing three binding sites for the cellular factor sp1. After incubation of labeled oligonucleotides, either unmodified or containing O6-MeG, with nuclear extracts obtained from different cell lines, gel retardation assays indicated that the presence of O6-MeG resulted in inhibition of binding of cellular factors to DNA sequences located in the promoter regions of genes. This inhibition was not the same for all modified oligonucleotides but dependent on the position in which O6-MeG was located. The results obtained indicate that alkylation of O6-guanine affects the binding of transcription factors and thereby possibly the regulation of genes expression.
Phase I and clinical pharmacological evaluation of aphidicolin glycinate.
The toxicity profile and the pharmacokinetics of aphidicolin glycinate, a water-soluble analogue of aphidicolin, have been evaluated in two consecutive phase I clinical studies. In the first study, aphidicolin glycinate was given by 1-hour infusion for 5 consecutive days, every 3 weeks (daily x 5 study); in the second study, which was planned on the basis of the pharmacokinetic information obtained in the previous study, the drug was given by 24-hour continuous infusion. Treatment was repeated every 3 weeks. In the daily x 5 study, the daily dose was escalated from 12 mg/m2 to the maximum tolerated dose of 2250 mg/m2. Local toxicity was dose limiting. Elimination half-life was 2 +/- 0.2 hours (mean +/- SE) with aphidicolin being undetectable 6-8 hours after the end of the infusion. In the 24-hour continuous-infusion study, the dose was escalated from 435 mg/m2 to the maximum tolerated dose of 4500 mg/m2. Local toxicity was dose limiting, while other toxic effects were absent. The experimentally determined concentrations at the steady state were in agreement with those predicted on the basis of the available pharmacokinetic data. The targeted concentration at the steady state of 3 micrograms/mL was achieved at doses greater than or equal to 3000 mg/m2. Twenty-four-hour continuous infusion is the recommended schedule for clinical evaluations of aphidicolin glycinate as the synchronizing agent or in combination with cisplatin.
Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase.
A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells.
Selective DNA interaction of the novel distamycin derivative FCE 24517.
N-Deformyl-N-(4-N-N,N-bis(2-chloroethylamino)benzoyl)distamy cin A (FCE 24517) is a novel cytotoxic and antitumor agent shortly to be investigated in phase I clinical trials. It was equally effective in inhibiting the growth of the murine L1210 line and of a subline (L1210/PAM) resistant to nitrogen mustards, whereas distamycin A was virtually inactive. The cellular uptake and retention of FCE 24517 and distamycin A were similar, thus excluding the possibility that this marked variation in cytotoxic activity was due to different intracellular concentrations of the two compounds. FCE 24517 did not appear to act as an inhibitor of macromolecule synthesis. As shown by radioactively labeled precursor incorporation only 24 h after drug treatment a significant inhibition of DNA synthesis was observed in L1210 or in L1210/PAM, when a marked proportion of cells was arrested in premitotic phase. FCE 24517 did not cause DNA breaks, DNA interstrand cross-links, or DNA-protein cross-links in L1210 cells exposed to active drug concentrations. A very low amount of radioactivity was found to be bound irreversibly to DNA in L1210 cells exposed for 1 h to [14C]FCE 24517. Using plasmid pBr322 DNA fragments in a modified version of the Maxam and Gilbert DNA sequencing technique we found no detectable binding of FCE 24517 to N-7-guanine (the major site of alkylation for classical alkylating agents), whereas some alkylations to adenine (presumably to N-3-adenine) were demonstrated. Thus it appears that FCE 24517 is a novel antitumor agent with a mode of action different from that of the drugs currently used in the clinic. In summary it is suggested that FCE 24517 acts by causing a few selective alkylations to adenines in the minor groove of DNA, although the precise base sequence necessary has yet to be elucidated.
Studies of the differentiation properties of camptothecin in the human leukaemic cells K562.
Camptothecin, a specific inhibitor of topoisomerase I, caused erythroid differentiation of the human leukaemia cell-line K562, as assessed by benzidine staining at 70 h recovery following a 60 min treatment of the cells. Differentiation was confirmed by increased levels of epsilon-globin and gamma-globin mRNA in the treated cells and was accompanied by down-regulation of c-myb mRNA. Synchronisation of K562 cells by non-cytotoxic doses of methotrexate increased the differentiation induced by camptothecin, without affecting the camptothecin-induced inhibition of cellular proliferation. Camptothecin induction of differentiation and inhibition of proliferation may occur by independent mechanisms.
The inhibition of supercoiling activity of DNA gyrase from Micrococcus luteus caused by rufloxacin (MF 934) and MF 961.
Explore the source record for details and available documents.
Concentrations of VP16 and VM26 in human brain tumors.
VP16 and VM26 were determined by high pressure liquid chromatography in intracerebral tumors, adjacent normal brain tissue, plasma and CSF samples from 24 patients given the two drugs before surgical resection of the tumor. The drugs were administered at doses of 100-150 mg/m2 as a 1-hour i.v. infusion, between 1.5 and 12 hours before surgery. Concentrations of VP16 ranged between 1.05 and 3.28 micrograms/g in tumors in the series of patients who received the drug 1.5-3 hours before surgery and between less than 0.05 and 1.12 micrograms/g in four patients who received the drug 9-13 hours before surgery. Tumor concentrations of VM26 also varied, ranging from less than 0.05 to 1.68 micrograms/g between 1.5 and 12 hours before surgery. VP16 and VM26 in the apparently normal brain tissue surrounding the tumor were low or undetectable except in one patient who had received radiotherapy, in whom we found 3.1 micrograms/g.
Natural killer (NK) and lymphokine activated killer (LAK) cell activity in patients (PTS) treated with flavone acetic acid (FAA).
Natural killer (NK) cell activity was measured in nine patients with different solid tumors treated with flavone acetic acid (FAA). In three of them, NK cell activity was significantly increased, in two instances even after repeated courses of treatment. A significant increase in lymphokine activated killer (LAK) cell activity was also observed in three of four patients after in vivo treatment with FAA and in vitro incubation of lymphocytes with recombinant Interleukin-2. We observed some inter-individual variability in drug clearance, but no apparent correlation between drug plasma levels (neither peak nor AUC values) and the effects on NK and LAK activity.
AACR special conference in cancer research. Membrane transport in multidrug resistance, development, and disease. American Association for Cancer Research.
Explore the source record for details and available documents.
Nerve cell death induced in vivo by kainic acid and quinolinic acid does not involve apoptosis.
We investigated whether in vivo excitotoxicity was mediated by a mechanism of programmed cell death called apoptosis. Neurotoxic doses of kainic acid (1.2 nmol) and quinolinic acid (120 nmol) were unilaterally injected in the dorsal hippocampus of anesthetized rats. Eight or 16 h later the animals were killed and DNA was extracted from the injected hippocampi. DNA from mouse thymocytes exposed to methylprednisolone (10(-5) M for 6 h at 37 degrees C) was used as a positive control of apoptotic cells. No typical 'ladder' of DNA fragments (multimers of approximately 200 Kb) which characterizes apoptosis was seen in hippocampal cells after toxic doses of kainic or quinolinic acid, as assessed by agarose gel electrophoresis. This suggests that hippocampal nerve cell death induced in vivo by the excitotoxins is not mediated by apoptosis.
In vitro and in vivo effects of cisplatin on the generation of lymphokine-activated killer cells.
Pretreatment of human peripheral blood lymphocytes (PBL) with cisplatin (CDDP) before in vitro culture with interleukin-2 (IL-2) inhibited the generation of lymphokine-activated killer (LAK) cells and strongly inhibited proliferation. This inhibition was dose dependent, was significant only at concentrations greater than 6 microM, and it required exposure to the drug for more than 1 hour. This period of IL-2 unresponsiveness was maximum at 6 hours, but was spontaneously recovered within 24-48 hours and was more rapidly restored by increasing dosages of IL-2. Because inhibition of the generation of LAK cells by CDDP was observed only at relatively high levels of exposure to the drug (greater than 6 microM for greater than 1 hr), it was important that we explore the in vivo significance of these findings. The peripheral blood lymphocytes from patients bearing ovarian adenocarcinoma collected 1 hour after an iv infusion of 50 mg of CDDP/m2 were not inhibited, compared with those collected immediately before therapy. Relatively high levels of exposure to CDDP are required for inhibition of the generation of new cytotoxic effectors, most likely because of its antiproliferative effect. These results may bear relevance to approaches involving the combined use of CDDP and IL-2-LAK.
Increase in topoisomerase-II-mediated DNA breaks and cytotoxicity of VP16 in human U937 lymphoma cells pretreated with low doses of methotrexate.
Pre-treatment with low, non-toxic concentrations (0.04 microM) of methotrexate (MTX) for 16 hr increased etoposide (VP16)-induced growth inhibition and cytotoxicity in the U937 human histiocytic lymphoma cell line. VP16 cytotoxicity was significantly potentiated when the drug was given for 2 hr immediately after MTX pre-treatment or between 2 and 4 hr or 4 and 6 hr after recovery from MTX pre-treatment. By 24 hr after recovery from MTX, no potentiation was evident. The increased cytotoxicity of VP16 was associated with an increase in drug-induced DNA breaks as assessed by the alkaline elution method after proteinase K digestion. The amount of DNA single-strand breaks (DNA SSB) increased when the drug was given 0, 2, and 4 hr after MTX pre-treatment. DNA SSBs induced by the drug between 6 and 24 hr after MTX pre-treatment were similar to those seen in cells without pretreatment. The amount of DNA double-strand breaks (DNA DSB) caused by VP16 increased significantly when the drug was given 4 hr after recovery from MTX pre-treatment. VP16-induced DNA DSBs were still higher 6 hr after MTX pre-treatment, but by 24 hr they were similar to those observed in MTX-untreated cells. Flow cytometric analysis showed that MTX pre-treatment was causing an accumulation of U937 cells at the G1-S boundary of the cell cycle. When MTX was removed, a wave of synchronization followed. Using Western blot electrophoresis and polyclonal antibodies to antitopoisomerase II, we found that MTX pre-treatment raised the cellular topoisomerase II content. Our findings suggest that the potentiation of VP16 cytotoxicity on U937 cells by low, non-toxic MTX pre-treatment is due to a larger fraction of S-phase cells containing a higher concentration of topoisomerase II, which is the putative target of VP16 action.
Synchronisation of cancer cell lines of human origin using methotrexate.
U937 human histiocytic lymphoma cell line and SW626 ovarian carcinoma line of human origin were synchronised using very low, nontoxic concentrations (0.04-0.08 microM for 16-24 h) of methotrexate (MTX) under standard culture conditions. Satisfactory synchrony was achieved to study S phase events. Various kinetic behaviours and biological properties of the synchronised cells are considered for characterisation of the system. MTX-synchronisation was compared with that induced by aphidicolin (APC) alone and by serum deprivation and APC. In some cancer cell lines MTX appears to be the best choice for obtaining highly synchronised cell populations without cytotoxicity or physiological perturbations.
DNA damage and cytotoxicity of mitoxantrone and doxorubicin in doxorubicin-sensitive and -resistant human colon carcinoma cells.
The effects of mitoxantrone (Mx) and doxorubicin (Dx) on cytotoxicity and DNA damage as assayed by alkaline elution were studied in two human colon adenocarcinoma cell lines sensitive (LoVo) and resistant (LoVo/Dx) to doxorubicin. Mx was more cytotoxic than Dx to LoVo cells and was partially cross-resistant in LoVo/Dx. In LoVo cells, Mx produced about 5 times more DNA single-strand breaks (DNA-SSB) than Dx, but both drugs caused an equal number of DNA double-strand breaks (DNA-DSB). In LoVo/Dx cells, the number of DNA-DSB was very low for both Dx and Mx, but DNA-SSB were about 20 times higher for Mx. In LoVo cells, the number of DNA-DSB and protein-associated SSB were similar at equitoxic concentrations. For LoVo/Dx, the partial cross-resistance of Mx might be explained by the much higher number of DNA-SSB produced by this drug.
Pharmacokinetics of 4-demethoxydaunorubicin in cancer patients.
The clinical pharmacokinetics of 4-demethoxy-daunorubicin was investigated in 28 cancer patients who received the drug orally. The majority of the patients were elderly (median age, 72 years). Nine of them also received an i.v. dose, and the bioavailability of the oral dose ranged between 9% and 39%. 4-Demethoxydaunorubicin peak levels were achieved 2-4 h after the oral dose in most patients. The drug was rapidly and extensively metabolized to 4-demethoxy-13-hydroxydaunorubicin, which is probably as active as the parent drug. The metabolite levels were much higher and longer lasting than the parent drug, suggesting that it may play an important role in the drug's pharmacological effects.
Flavone acetic acid antitumour activity against a mouse pancreatic adenocarcinoma is mediated by natural killer cells.
Flavone acetic acid (FAA) is one of the most active antitumour agents against mouse solid tumours. A number of reports favour the hypothesis that FAA could behave as a biological response modifier; in fact FAA stimulates natural killer (NK) cells, induces secretion of type I interferon and synergizes with interleukin-2 to increase NK/lymphokine-activated killer (LAK) activity in vivo. However, there is no conclusive evidence that the antitumour activity of FAA is mediated via the modulation of NK/LAK cells. The present study was designed to evaluate whether the reported activation of NK cells is instrumental in FAA antitumour activity. FAA (180 mg/kg, i.v. on days 3, 7 and 11 after tumour implant) was significantly effective in inhibiting the subcutaneous growth of the pancreatic adenocarcinoma PAN/03 in C57/Bl mice. After 132 days the number of tumour-free survivors was 36%, whereas in the control group receiving no treatment, or in the group of mice treated with 10 micrograms/mouse of alpha-asialo-GM1 the value was only 0 or 6.7%, respectively. The combination of FAA and alpha-asialo-GM1 resulted in only 6% tumour-free mice. In parallel experiments, splenocytes and peritoneal cells from C57/B1 mice were tested in a standard cytotoxicity NK assay. While animals treated with FAA showed a significant increase in NK activity, those injected with alpha-asialo-GM1 had very low levels, and the combined treatment of FAA and alpha-asialo-GM1 resulted in a lower or similar NK activity compared to that in untreated mice. The fact that the abrogation of the NK-stimulating effect of FAA is accompanied by a lack of anti-tumour activity indicates that, at least in this experimental model, FAA is likely to act via an immunomodulatory mechanism.