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Biomedical subjects

M D'Incalci

Publications and source records attributed to M D'Incalci.

At least 217 records · Page 12Linked to original sources
PubMed1989

Flow cytometric analysis of DNA content in human ovarian cancers.

A total of 155 samples from 101 patients with ovarian cancer were investigated using flow cytometry to evaluate the DNA index and the percentage of cells in the various cell cycle phases. Thirty-four samples were DNA diploid tumours, while the other 121 were DNA aneuploid tumours. The DNA index was very stable in different sites and over time in the same patient. Tumour stage and ploidy were significantly associated: stages III and IV tumour stage were more likely to be DNA aneuploid. Patients with residual tumour size at first surgery greater than 2 cm had a significantly larger number of DNA aneuploid than DNA diploid tumours. The DNA index was also related to the degree of differentiation of the tumours. The percentage of cells in the S phase of the cell cycle was significantly higher in DNA aneuploid and in poorly differentiated tumours than DNA diploid and well differentiated tumours. Multivariate analysis using the Cox model showed that the DNA index and the percentage of cells in S phase were not independent prognostic variables in this study. Prospectively collected data should be accumulated before assigning the DNA index an important role as a biological prognostic factor in ovarian cancer.

DNA, Neoplasm↗
PubMed1989

Problems in the design of anticancer agents.

Theoretically, two predominant paths for obtaining more selective anticancer agents may be envisaged. These are: (a) to make compounds which distribute only or preferentially in cancer cells; (b) to make compounds that are able selectively to kill or to differentiate cancer cells. Although in the last two decades research into new anticancer drugs has not produced satisfactory results, there is solid ground on which novel strategies can be developed, mainly based on a much greater biological knowledge of human tumours. This article does not review all the possible approaches that may be followed, but simply discusses some ideas and problems mainly taken from the current research of our laboratory.

Animals↗
PubMed1988

Intracellular doxorubicin concentrations and drug-induced DNA damage in a human colon adenocarcinoma cell line and in a drug-resistant subline.

The mechanisms of resistance to doxorubicin (DX) were investigated using a human colon adenocarcinoma cell line (LoVo) and a subline approximately 30 times less sensitive to doxorubicin. LoVo and LoVo/DX were similar in terms of DNA and protein content, cell volume, duration of S phase and the generation time, and proportion of cycling cells. LoVo/DX showed cross-resistance to other anthracyclines, to vinca alkaloids, epipodophyllotoxin derivatives, 4'-(9-acridinylamino-methanesulfon-m-aniside) and actinomycin D. LoVo/DX was equally sensitive to melphalan and showed collateral sensitivity to cis-platinum and 1-beta-D-arabinofuranosylcytosine. On exposing LoVo and LoVo/DX to 1.25 and 40 micrograms/ml DX respectively, for 4 hr, similar DX intracellular concentrations were reached in the two cell lines. In these treatment conditions protein associated DNA-single strand breaks or DNA-double strand breaks, assessed by alkaline elution methods were only slightly less in LoVo/DX than in LoVo cells. In LoVo/DX cells, however, DNA breaks disappeared very quickly after drug removal whereas they persisted longer in LoVo cells. This persistance is probably related to the much slower DX efflux from LoVo than LoVo/DX. When verapamil was combined with DX it inhibited the rapid DX efflux from LoVo/DX and reversed the resistance in this cell line, but it had no significant activity on LoVo cells. Verapamil also increased DX-induced DNA-single strand breaks and DNA-double strand breaks in LoVo/DX cells, but not in LoVo cells.

Adenocarcinoma↗
PubMed1988

Doxorubicin induces the acetylation of histone H1 in a human colon cancer cell line (LoVo/DX) selected for resistance to the drug, but not in the sensitive parental line (LoVo).

The effect of doxorubicin (DX) treatment on H1 synthesis and acetylation was studied in two human colon adenocarcinoma cell lines, sensitive (LoVo) and resistant (LoVo/DX) to this drug. Histone variants were resolved by a high resolution two-dimensional gel electrophoresis system coupled to fluorography for the detection of radioactive incorporation. The relative synthesis of H1.4 and H1.5 variants was slightly reduced by DX. This is probably related to the inhibition of DNA synthesis consequent to drug treatment. The main effect is that DX induces the acetylation of H1 isoproteins in the LoVo/DX resistant line but not in the parental line, which is 30 times more sensitive to anthracyclines. The different behavior of the two cell lines cannot be attributed to different cellular drug retention since the DX doses chosen (1.25 for LoVo and 40 micrograms/ml for LoVo/DX cells) correspond to similar intracellular drug concentrations. H1 acetylation persisted after exposure to cycloheximide in DX treated LoVo/DX cells, indicating that it is a postranslational event. The induction of H1 acetylation appears rather specific since no increase was found in 3H-acetate incorporation on the total cellular TCA-precipitable fraction. In addition DX treatment did not modify the acetylation of core histones in either LoVo or LoVo/DX cell lines.

Acetates↗
PubMed1988

Comparison of histone variant synthesis in human lymphocytic leukemia cells and in normal lymphocytes.

The synthesis of core histone variants and of histone H1 variants was determined in fresh leukemic cells of eight patients with leukemia [seven acute lymphoblastic (ALL) and one chronic lymphocytic (CLL)], in normal lymphocytes from healthy donors or from ALL patients in complete remission. Histone variant synthesis was evaluated by incubating cells with [14C]Lys and [3H]Arg in medium without Lys and Arg and then by two-dimensional polyacrylamide gel electrophoretic separations (acetic acid-urea-Triton x-100 acetic acid-urea-hexadecyltrimethylammonium bromide for core histone variants; sodium dodecyl sulfate/acetic acid-urea-hexadecyltrimethyl ammonium bromide for H1 variants). As previously reported, quiescent lymphocytes and lymphocytes stimulated with phytohaemagglutinin (PHA) showed clearcut changes in the proportions of synthesis of core histone variants and H1 variants. Leukemic lymphocytes freshly obtained from blood showed a pattern of core histone synthesis and H1 synthesis intermediate between that of quiescent and PHA-stimulated lymphocytes; this is probably due to the presence of a mixture of resting and growing cells. When leukemic cells were stimulated to grow by mitogens, the pattern of core histone and H1 variant synthesis was similar to that in mitogen-stimulated normal lymphocytes. Histone variants whose synthesis is associated with the S-phase were not synthesized in leukemic cells treated with the DNA synthesis inhibitors hydroxyurea and 1-beta-D-arabinofuranosylcytosine (Ara-C). The pattern of acetylation of histone H4 was also apparently similar in leukemic cells and normal lymphocytes. The radioactivity associated with the ubiquitinated forms of H2A increased in nongrowing lymphocytes and in leukemic cells treated with DNA synthesis inhibitors whereas they decreased after mitogenic stimulation. Variability was wide in the synthesis of ubiquitinated H2A in different cases of leukemia. The only clear-cut difference between leukemic cells and normal lymphocytes was that leukemic cells from ALL patients, but not lymphocytes from normal donors or from ALL patients in complete remission, synthesized appreciable amounts of H1 degrees, increasing after hydroxyurea/Ara-C treatment and decreasing after PHA-stimulation. In leukemic cells from a CLL patient H1 degrees synthesis was undetectable.

Acetylation↗
PubMed1988

Human ovarian tumors in primary culture: growth, characterization and initial evaluation of the response to cis platinum treatment in vitro.

A novel cell culture system is reported for the growth of ovarian tumors. Two approaches were developed to isolate tumor cells, one for ovarian carcinomas and the other for benign cystomas or borderline cystadenomas, which yield virtually pure tumor-cell clusters. The plating efficiency exceeded 10% in approximately 80% of the processed surgical specimens. Cells grown in a newly developed KOV medium (a modification of MCDB 151 supplemented with 6 defined growth factors and a moderate amount of FBS) had an average growth rate of 0.23 population doublings/day. Primary tumor-derived cultures, including those derived from cystomas, were analyzed by flow cytometry demonstrating a DNA heteroploid content in 55% of the cases. The neoplastic origin of the cells in culture was further confirmed by 3 monoclonal antibodies (OC125; MOv2; MOv19) with high specificity against epithelial ovarian malignancies. Cultures were tested with cis-DDP to determine their suitability for pharmacological studies. Exposure to the drug (from 10 to 80 microM for 1 hr) resulted in variable cell-killing responses, and the prominent effect on cell-cycle progression in primary cultures was a prolonged arrest in S phase. The formation and persistence of DNA-ISC caused by an exposure to 40 microM cis-DDP for 1 hr was studied by alkaline elution in 6 different tumor-derived cultures. DNA-ISC equivalents were highest between 9 and 24 hr after treatment and were repaired only to a limited extent within 48 hr of recovery time. The present study confirms the usefulness of this culture system for pharmacological studies of active chemotherapeutic agents against human ovarian tumors.

Antibodies, Monoclonal↗
PubMed1988

Accumulation of DNA strand breaks in cells exposed to methotrexate or N10-propargyl-5,8-dideazafolic acid.

N10-Propargyl-5,8-dideazafolic acid (CB 3717), a new antifolate which directly inhibits thymidylate synthase and which is now under early clinical investigation, was compared with methotrexate (MTX) for its antiproliferative activity and mode of action on M14 human melanoma cell line and NIH/3T3 murine fibroblasts transfected with human c-Ha-ras oncogene (NIH/3T3R). CB 3717 was as active as MTX on both cell lines in inhibiting colony formation, but 20-100 times less potent. After 24 h of exposure both drugs caused an accumulation of cells in the G1 phase of the cell cycle, probably because of inhibition of DNA synthesis and blockage at the G1-S boundary. In NIH/3T3R treated for 16 h with 2 microM MTX or 200 microM CB 3717, we found DNA single-strand breaks amounting to approximately 130 and 140 rad equivalents, respectively, and a considerable number of DNA double-strand breaks, far more than expected if they had been the result of the proximity of single-strand breaks on the two complementary DNA strands. No DNA-protein cross-links were detected. When cells were incubated in drug-free medium for 8 h, there was a further accumulation of single-strand breaks, possibly due to the effects of the drug retained intracellularly as polyglutamyl derivative. Simultaneous treatment with 1.77 microM cycloheximide prevented DNA damage produced by both drugs. Thymidine (10 microM), renewed in the culture medium every 24 h, also prevented DNA damage and cytotoxicity. Since after 16 h treatment with MTX or CB 3717 cells were completely viable, as assessed by [3H]thymidine release, trypan blue exclusion test, and 51Cr release, DNA damage appears to be an early event preceding cell death and may be a feature of the killing ability of the drugs. The involvement of a protein in the formation of DNA breaks is suggested by the fact that when protein synthesis was inhibited with cycloheximide DNA damage was no longer seen.

Cell Cycle↗
PubMed1988

DNA damage, cytotoxic effect and cell-cycle perturbation of Hoechst 33342 on L1210 cells in vitro.

This study was designed to evaluate the effects of vital dye Hoechst 33342 (HO 33342), at concentrations used to obtain a good DNA histogram resolution, on DNA integrity, cell growth, and cell-cycle phase distribution of L1210 cells. HO 33342 exposure for 2 h, at 37 degrees C produced DNA single-strand breaks as assessed by the method of alkaline elution. DNA single-strand breaks were concentration dependent (in the range .5-5 micrograms/ml) and increased significantly when HO 33342 (0.5-1.5 micrograms/ml) was associated with exposure in a flow cytometer to U.V. laser beam illumination. HO 33342 produced a cytotoxic effect on cell growth even at the concentration of 0.5 microgram/ml--a concentration ten-fold smaller than those required to obtain a good DNA histogram resolution. HO 33342 produced a severe block of the cells in the G2-M phase of the cell cycle already evident 24 h after stain exposure and continuing up to 144 h after start of recovery. A new polyploid cell population (with a 4 c DNA content) not present in the unstained cells was already evident 24 h after dye exposure. The data shown in the present paper would imply caution in using sorted cells stained with HO 33342 dye for biological, biomedical, and pharmacological studies.

Animals↗
PubMed1988

Dose-dependent pharmacokinetics of flavone acetic acid in mice.

The pharmacokinetics of the novel anticancer agent, flavone acetic acid (FAA) were investigated in Balb-c mice treated with i.v. doses of 100 mg/kg or 300 mg/kg, using an HPLC assay. The kinetics of disappearance from plasma was monoexponential and dose-dependent. After 100 mg/kg, the plasma peak level was 250 +/- 11 micrograms/ml, t1/2 was 0.5 h, and AUC was 309 micrograms/ml per h. After 300 mg/kg, the plasma peak level was 710 +/- 57 micrograms/ml, t1/2 was 2.1 h, and the area under the curve (AUC) 1771 micrograms/ml h. Mouse plasma protein binding of FAA was about 70%. As is the case with plasma, in all tissues analyzed, the FAA-AUC values were disproportinately greater after 300 mg/kg than after 100 mg/kg. The highest drug concentrations were found in the liver and small intestine; concentrations were intermediate in lung, heart, and spleen, and lowest in brain. Less than 5% of the FAA dose was eliminated as unchanged drug in the stool. Total excretion of FAA as unchanged drug in the urine collected up to 96 h after drug treatment corresponded to 75% and 60% of the i.v. doses of 100 and 300 mg/kg, respectively. A minor fraction of FAA dose, corresponding to 1% and 6% of the two doses, was eliminated in the urine as a FAA glucuronide or sulfate.

Animals↗
PubMed1988

Lack of effect of cisplatin on i.v. L-PAM plasma pharmacokinetics in ovarian cancer patients.

Melphalan (L-PAM) pharmacokinetics were investigated in nine ovarian cancer patients before and after cisplatin (DDP) treatment. When L-PAM was given 24 h before DDP, the elimination half-life (t 1/2 beta), plasma clearance (Clp), and volume of distribution (Vd beta) of L-PAM were, respectively: 46.4 +/- 6.7 min, 20.5 +/- 3.7 l/m2, and 306.8 +/- 34.4 ml/min per square meter. When L-PAM was inoculated 24 h after DDP, t 1/2 beta, Clp, and Vd beta were 47.5 +/- 6.3 min, 20.4 +/- 2.8 l/m2, and 322.0 +/- 54.1 ml/min per square meter. Thus, DDP pretreatment does not significantly affect L-PAM pharmacokinetics. Regression analysis showed a significant correlation between the L-PAM elimination rate constant (beta) and renal function assessed by creatinine clearance. One patient who received this sequence of treatment for six courses showed a threefold decrease of L-PAM Clp after the last treatment. The reported high myelotoxicity of the combination of DDP and L-PAM when DDP was given 24 h before L-PAM cannot be attributed to DDP-induced changes in L-PAM kinetics but might to some extent be related to a loss of renal function consequent to many courses of treatment.

Adult↗
PubMed1988

Phase I study of the antifolate N10-propargyl-5,8-dideazafolic acid, CB 3717.

The thymidylate synthase (TS) inhibitor CB 3717 was administered intravenously to 24 adult patients as a single bolus repeated every 3-4 weeks. The doses were escalated from 50 to 400 mg/m2. At the highest level, hydration and urinary alkalinization were routinely performed. A greater than 20% decrease of the creatinine clearance value occurred in 35% of the cycles performed at 400 mg/m2, a dose which could be recommended for phase II studies. Hepatic toxicity, represented by an increase of the glutamic pyruvic transaminase (GPT) plasma levels, occurred in 70% of the patients after the first cycle. GPT peak levels were neither related to the dose nor to the peak drug plasma concentrations or AUC values in the dose range from 225 to 400 mg/m2. Malaise, reported after 46% of the cycles, was the most disturbing side-effect and its occurrence was statistically correlated with the degree of elevation of GPT. A suggestion of antitumor activity was reported for dosages of 300 and 400 mg/m2 in three patients with ovarian cancer refractory to cisplatin. Further clinical evaluations of TS inhibitors rely on the development of more water soluble and less hepato- and nephrotoxic agents.

Adult↗