Franca Majone

Human T-Cell Leukemia Virus Type 1 Tax and Cell Cycle Progression: Role of Cyclin D-cdk and p110Rb

ABSTRACT Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16INK4a, thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16INK4a, Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16INK4a.

Genetic effects of chromium compounds

Seven different test systems were utilized to investigate the genetic activity of chromium compounds: infidelity of DNA replication in vitro by DNA pol alpha from calf thymus, damage of DNA detected by alkaline elution in treated mammalian cells or in DNA purified and treated in vitro, DNA repair synthesis in mammalian cells in vitro detected by autoradiography or scintillation counting after labelling with [3H]dThd, gene mutations in the Salmonella typhimurium Ames test, gene mutations (6TG resistance) in cultured hamster cells, sister-chromatid exchanges in different rodent cell cultures, and transformation to anchorage-independent growth of hamster cells in vitro (soft-agar assay). Potassium dichromate and chromium chloride were used as water-soluble Cr(VI) and Cr(III) salts. Several reference mutagens (EMS, MMS, MMC, 4NQO) were included in the single tests as positive controls. Cr(VI) was active in all the tested systems, except in the induction of DNA damage and DNA repair synthesis in cultured cells. Cr(III), on the other hand, was absolutely inactive unless a direct interaction with purified DNA was permitted by the test conditions. The relevance of data from the various tests to the understanding of the mechanisms of the genotoxic activity of chromium is discussed. Effects other than the direct interaction of Cr(III) with DNA are inferred, which can cause infidelity of the DNA polymerase functions.

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.

Persistence of micronuclei in the marine mussel, Mytilus galloprovincialis, after treatment with mitomycin C

The frequency of micronuclei induced by mitomycin C (MMC) in cells of the gill tissue of the marine mussel, Mytilus galloprovincialis Lmk., was determined over a long period (up to 40-52 days) following treatment. Two doses of MMC (0.5 X 10(-7) and 10(-7) M) were tested at 13 degrees C and 23 degrees C, temperatures representative of the winter and summer thermic conditions of the Mediterranean Sea. In all cases, the frequency of micronuclei was significantly increased by MMC and declined after treatment until it reached a plateau level, significantly higher than the control value. This persisted for a very long time. The frequency of micronuclei induced by a second treatment with MMC performed on the 28th day, did not differ significantly from that produced by the first treatment at the same dose. Temperature did not influence the pattern of the described phenomena to a significant extent. The reason for the persistence of an increased frequency of micronuclei is discussed, and a system is proposed for evaluating the genotoxicity of water pollutants present long before sampling.

Mechanisms of chromium toxicity in mammalian cell cultures

Our observations about the cytotoxic and cytogenetic effects of hexavalent and trivalent chromium compounds in mammalian cells cultured in vitro are reviewed. Additional data concerning the induction of chromosomal aberrations and sister chromatid exchanges, the inhibition of nucleic acid and protein synthesis, the interference with nucleotide metabolism, and the modification of membrane-linked enzyme activity are reported. A possible mechanism of chromium action is proposed.

Tax Protein of Human T-Lymphotropic Virus Type I Triggers DNA Damage

Previous findings indicated that in vitro HTLV-I-infected cells are highly susceptible to spontaneous and chemically induced DNA-damage. To further study the role of different virus gene products in inducing chromosome abnormalities, MOLT-3 cells were transiently transfected with a tax expressing plasmid (pTax), and assayed for genetic damage by the micronucleus test. We found that pTax-transfected cells not only had a statistically higher baseline micronucleus value than non-transfected control cells, but also were more susceptible to Mitomycin C (MMC)-induced DNA damage. Furthermore, the use of human serum containing anti-kinetochore antibodies disclosed that tax enhances the clastogenic effect of MMC. No increase in total micronucleus frequency was observed when MMC treatment preceded pTax transfection, thus suggesting that the micronucleus increase might not be due to the additive effect of tax and MMC. These findings indicate that the viral tax protein could play an important role in inducing the chromosome damage frequently observed in HTLV-I-infected cells.

Sister-chromatid exchange in developing eggs of Mytilus galloprovincialis Lmk. (bivalvia)

Abstract A simple and rapid experimental technique for detecting SCE in developing eggs of Mytilus galloprovincialis Lmk. (Bivalvia) was set up. Eggs, fertilized in the laboratory, were treated with nitrilotriacetic acid (NTA), HgCl 2 and HgCl 2 plus NTA (1 : 1). Results confirm the high genotoxicity of mercury, indicating an absence of activity by NTA and a lack of interaction between NTA and the metal. Eggs from mussels collected in two stations characterized by different levels of sea-waste and hydrocarbon pollution presented a significant difference in their SCE frequencies. This indicates that a genetic effect can also be produced in gametes. This system may be used for laboratory studies as well as for monitoring the environmental genotoxicity of marine pollutants.

Frequencies of micronuclei detected on Mytilus galloprovincialis by different staining techniques after treatment with zinc chloride

The frequencies of micronuclei induced by ZnCl2 and detected on the gill tissue of the marine mussel Mytilus galloprovincialis with different staining techniques (acridine orange, gallocyanin chromallum, Feulgen, Giemsa) were compared. At least in the used system, the Feulgen and gallocyanin chromallum methods gave a frequency of micronuclei significantly lower than that obtained with the acridine orange and Giemsa techniques. No significant difference between the frequencies obtained with acridine orange and Giemsa was shown. So, though the acridine orange is surely the method which provides the more reliable data, in environmental screening works the Giemsa technique may be more suitable for its simplicity.

Effects of Potassium Dichromate on Mitosis of Cultured Mammalian Cells

SUMMARYWe have analyzed the effects of a soluble Cr6+ compound (potassium dichromate) in a human cell line (HEp), taking into account: a) the changes of mitotic index;b) the changes of the frequency of the different mitotic phases;c) the induction rates of the main caryological and cytological alterations.Our experiments indicate that dichromate induces: 1) a blockage of the cells which are in mitosis at the time of treatment, which takes place at metaphase and anaphase;2) an early mitotic inhibition on the cells which are in the G2 phase of the cell cycle;3) a recovery of cell division, after the mitotic inhibition, apparently accompanied by a partial cell synchronization;4) a late mitotic blockage when cell division recovers, which occurs mainly at metaphase. Such blockage is related to several caryological and cytological alterations; sticky or lagging unoriented chromosomes in the metaphase plate, chromosome bridges at anaphase and telophase, cytoplasmic bridges at interphase;5) a large proportion of ...

Induction of micronuclei by mitomycin C and colchicine in the marine mussel Mytilus galloprovincialis

The frequencies of micronuclei (MNi) in the gill cells of the mussel Mytilus galloprovincialis were determined over a long period (up to 28 days) following a 48-h treatment with colchicine. The frequency of MNi at the end of treatment was significantly higher than in controls and 24 h later it had increased even more. After this period, the frequency of MNi rapidly declined until a plateau level was reached on day 2-3, which was significantly higher than the control baseline level, and persisted until the end of the experiment (28th day). In the same cell system we previously reported a persistence of an increased frequency of MNi after treatment with mitomycin C (MMC) (Majone et al., 1987). In order to establish the origin of MNi, the difference between their size distribution in MMC- and colchicine-treated animals was determined at the end of treatment as well as during the plateau phase. The difference was statistically significant (P less than 0.001) at the end of treatment, the MNi induced by MMC being smaller than those induced by colchicine. However, the difference during the plateau phase was not statistically significant. Human CREST antikinetochore fluorescent antibodies reacted with chromosome centromeres of Mytilus and were applied to gill cells at the end of a 48-h treatment with MMC or colchicine. About 60% of the MNi induced by colchicine but only 30% of those produced by MMC reacted positively with the fluorescent antibodies. This result indicates that the majority of MNi observed at the end of a 48-h treatment with MMC or colchicine originate, respectively, from acentric chromosome fragments and from whole lagging chromosomes.