The effect of AZT and chloroquine on the activities of ricin and a saporin-transferrin chimeric toxin.

This study deals with the combination of chloroquine (CQ, an anti-malaric drug) and 3'-azido-3'-deoxythymidine (AZT, anti-human immuno-deficiency virus (HIV) drug) with a chimeric toxin (TS) obtained by chemical linking of saporin (a ribosome inactivating protein from the plant Saponaria officinalis) and human transferrin, in the intoxication of the human chronic myeloid leukaemia cells (K562). Our data demonstrate that AZT, at concentrations comparable to those reached in the blood of HIV-infected patients under pharmacological treatment with this drug, can increase the toxicity of TS in cooperation with CQ inducing an increased effect on protein synthesis in K562 cells ( approximately 50% inhibition of protein synthesis for TS alone, and TS with AZT and approximately 70% with both AZT and CQ). Furthermore, pre-treatment of cells with AZT alone can induce an increase of apoptosis in K562 cells intoxicated with TS. By comparing data obtained with the model toxin ricin, we get indications that the two toxins partially differ in their intracellular routes, also suggesting that chimeric constructs containing ricin-like toxins (i.e. immunotoxins) could be coupled with the use of common and cheap drugs for the treatment of cancer in HIV-infected patients.

Purification and partial characterization of an alpha-2,8-sialyltransferase from human erythroleukemia K562 cells.

An alpha-2,8-sialyltransferase (ST8), the enzyme involved in the biosynthesis of polysialic acid chains, has been purified and partly characterized from undifferentiated human erythroleukemia K562 cells. Purification, based on a key step of affinity chromatography utilizing immobilized colominic acid, was greater than 1000-fold. The enzyme molecular weight determined by SDS-PAGE was estimated to be about 40 kDa, in good agreement with literature data. For the determination of the main kinetic parameters (Vmax and K(M)), fetuin turned out to be the unique substrate acceptor. In fact, other compounds such as asialofetuin, transferrin, alpha1-acid glycoprotein, and G(M3), routinely used to explore the different ST8 isoforms' activities, did not serve as substrate acceptors. In all cases, contrary to the routinely adopted protocol where a radioactive substrate donor is employed, for our purpose a non-radioactive, fluorescent substrate donor such as cytidine-5'-monophospho-9-(3-fluoresceinylthioureido)-9-deoxy-N-acetyl-neuraminic acid (CMP-9-fluoresceinyl-NeuAc) was used. Thus, under our experimental conditions, by using fetuin, data reported in a typical Lineweaver-Burk plot gave a Vmax value of about 4 nkatal/mg of protein and a K(M) value around 0.61 mM. Just as with the estimated molecular weight, these kinetic data were also in good agreement with those already reported for the ST8 purified from human neuroblastoma CHP-134 cells. In particular, in both cases, Vmax values were almost similar (4 nkatal/mg of protein for our ST8 purified from K562 cells and 4.35 nkatal/mg of protein for ST8 purified from CHP-134 cells); conversely, the K(M) value we found was about 3.25-fold lower than that found by Stoykova and Glick (0.61 mM vs. 2 mM). Then, although our purification was lower than that obtained by Stoykova and Glick (1080-fold vs. 2910-fold), the enzyme we purified showed a greater apparent affinity.

Evidences that zidovudine (AZT) could not be directly responsible for iron overload in AZT-treated patients: an in vitro study.

Zidovudine (3'-azido-3'-deoxythymidine or azidothymidine, AZT) has been the first antiretroviral agent approved for clinical use, and it is still currently used in combination therapy of human immunodeficency virus (HIV) infection. On the basis of increasing clinical reports and in vitro studies, a strict correlation between AZT treatment of HIV positive patients and both the development of anemia and iron overload have been in evidence over the last few years. In this report, we have examined some features of zidovudine to better assess a likely implication of this drug in iron overload. For this purpose, we first determinated the iron chelating ability of both AZT and some of its phosphorylated derivatives in solution. The iron chelating ability of AZT toward the intracellular 'chelatable' iron pool was also evaluated. Finally, we investigated the effect of AZT on both iron and transferrin uptake. Our findings indicate that AZT per se cannot be directly responsible for the development of the iron overload found in human or animal models, for which other possible mechanisms are claimed to be involved.

3'-Azido-3'-deoxythymidine reduces the rate of transferrin receptor endocytosis in K562 cells.

K562 cells, exposed for at least 24 h to 5 microM 3'-azido-3'-deoxythymidine (AZT), gave rise to an overall increase in the number of cell surface transferrin binding receptors (18-20%). This effect was ascertained either with binding experiments by using 125I-transferrin and with immunoprecipitation by using a specific monoclonal antibody against the transferrin receptor. At higher AZT concentrations (20 and 40 microM), a further increase was found, that is, up to 23% by binding experiments and up to 110% by immunoprecipitation. However, Scatchard analysis of the binding data indicated that although the number of cell surface transferrin receptors increased, the affinity of transferrin for its receptor did not change (Ka=4.0x108 M). Surprisingly, immunoprecipitation of total receptor molecules showed that the synthesis of receptor was not enhanced by the drug treatment. The effect of AZT on transferrin internalization and receptor recycling was also investigated. In this case, data indicated that the increase in the number of receptors at the cell surface was probably due to a slowing down of endocytosis rate rather than to an increased recycling rate of the receptor to cell surface. In fact, the time during which half the saturated amount of transferrin had been endocytosed (t1/2) was 2.15 min for control cells and 3.41, 3.04, and 3.74 min for 5, 20, and 40 microM AZT-treated cells, respectively. Conversely, recycling experiments did not show any significant differences between control and treated cells. A likely mechanism through which AZT could interfere with the transferrin receptor trafficking, together with the relevance of our findings, is extensively discussed.

Sister chromatid exchange (SCE) rates in human melanoma cells as an index of mutagenesis.

Melanomas are highly clonogenic. Genetic variability and polymorphism of tumour cell populations have been reported. However, no direct evidence of mutator activity as a source of genetic polymorphism for melanoma cells has been described. Some intermediates of melanin synthesis are cytotoxic and genotoxic and their mutagenic power has been described. We show here that the rate of sister chromatid exchange (SCE) of the line of human melanoma cells used varies with the concentration of the melanin precursor L-tyrosine, in the culture medium. An increase of melanin synthesis results in increased SCE rates. The highest values of SCEs are found in melanotic melanoma cells compared with the amelanotic ones. Indeed we present evidence that melanoma cells show higher levels of SCE when compared with normal human lymphocytes, and to the SCE frequencies derived from the literature on the lymphocytes of familial malignant melanoma, sporadic malignant melanoma patients and the lymphocytes of relatives of familial and sporadic melanoma patients.

Ultrastructural and phenotypic characterization of CABA I, a new human ovarian cancer cell line.

We have established an ovarian cancer cell line (CABA I) from ascitic fluid obtained from a patient with papillary adenocarcinoma of the ovary prior to drug treatment. The epithelial origin of the cell line was confirmed by morphology and by immunofluorescence analysis using anticytokeratin antibodies. Ultrastructural analysis revealed a very irregular membrane surface and a clear cytoplasm rich in electron-lucent vesicles. CABA I cells grow rapidly in culture (doubling time 18 h) in an anchorage-independent manner. Exogenously added beta-estradiol and epidermal growth factor (EGF) treatments did not influence cell growth rate. FACS analysis to determine the phenotypic profile of tumor-associated antigen, membrane receptor, and adhesion molecule expression indicated that the cell line was positive for different members of the c-erbB family, for alpha 6 and beta 1 integrin receptors, and intensively positive for HLA class I antigens and the folate receptor. Molecular characterization revealed no mutations for c-myc and c-k-ras genes, but did detect an exon 5 mutation in the p53 gene. CABA I cells grew poorly as heterotransplants in nude mice, and tumors showed long latency periods. Because early (15-20) and late (55-60) passage cells maintain the same growth and phenotypic characteristics, the CABA I cell line might provide a good in vitro model system to investigate the cellular and molecular events involved in ovarian carcinogenesis.

High resolution cytogenetic study in schizophrenia.

A high resolution cytogenetic study was performed on forty unrelated schizophrenic patients defined by DSM-III-R criteria. We have not included cases with mental retardation, severe dysmorphic features or other characteristic symptoms of chromosomal syndromes in our analysis. No recognizable chromosomal abnormality was found.