Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.

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 p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), 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 p16(INK4a).

Studies on the mechanisms of HTLV-I leukemogenesis.

The factors that regulate low viral expression and long latency after HTLV-I infection are poorly understood. To study the possible mechanisms involved in the regulation of gene expression and cell transformation, we studied whether (1) methylation could play a role in viral transcription, and (2) tax product could favor chromosomal instability. The results indicate that methylation of HTLV-I LTRs blocks their transcriptional activity and that tax protein triggers DNA damage.

Chromosome damage induced in cord blood T-lymphocytes infected in vitro by HTLV-I.

Experiments were carried out to investigate whether the human T-lymphotropic virus type I (HTLV-I), alone or in combination with a chemical mutagen such as mitomycin C (MMC), has the capacity to damage host chromosomes. Cord-blood T lymphocytes (CBL) were infected by co-cultivation with lethally irradiated HTLV-I-producing cells. Infected and immortalized CBL were then studied for frequencies of sister chromatid exchanges (SCE), chromosome breaks and micronuclei. HTLV-I-infected cells had statistically higher baseline SCE, chromosome aberrations and micronucleus values than the uninfected control CBL. While MMC treatment further augmented these values both in control and in infected lymphocytes, the latter did not show dose-related increases, most likely because of the more pronounced MMC-induced delaying effect on cell progression to mitosis. In view of similar previous observations in mouse lymphocytes carrying the Moloney murine leukemia virus, it is suggested that expression of a common retrovirus gene product, such as the pol endonuclease, might be responsible for the cytogenetic abnormalities observed. In addition to the IL-2 autocrine loop, the direct induction of chromosome damage by HTLV-I in target lymphocytes may be related to the pathogenesis of malignancies associated with HTLV-I infection.

Cytogenetic Effects Induced by Cytochalasin B in Chinese Hamster Ovary Cells.

We determined the kinetics of the induction of chromosomal aberrations and micronuclei (MN) by mitomycin C (MMC, 0.1 &mgr;g/ml) in Chinese hamster ovary (CHO) cells treated with cytochalasin B (Cyt-B, 3 &mgr;g/ml). In cells treated with Cyt-B as well as with Cyt-B plus MMC the highest yield of binucleated cells was obtained 24 h after treatment. After 40 h of treatment with Cyt-B the frequency of MN in binucleated cells was significantly higher than that observed at previous times in the same cultures as well as in controls. In cultures treated with MMC the frequency of MN increased with time, reaching the highest value at 24 h. The frequency of chromosomal aberrations was also significantly higher in cells treated both with Cyt-B and Cyt-B plus MMC than in controls and exceeded that of MN in parallel cultures. These data confirm the capacity of MMC to induce chromosomal alterations in mammalian cells; in particular they indicate that Cyt-B is able to induce cytogenetic effects in CHO cells. Using immunofluorescence microscopy, after reaction with CREST antikinetochore antibodies, we found that in cells treated with Cyt-B or Cyt-B plus MMC the frequency of MN without kinetochore was, respectively, about 70 and 85%, indicating that under our experimental conditions MN originate mainly from acentric chromatid fragments. Present data suggest that the method based on the blockage of cytokinesis by Cyt-B normally used in the MN assay should be reconsidered. Copyright 1995 S. Karger AG, Basel

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.

Biological properties of some benzopsoralen derivatives.

The biological activity of some benzopsoralen derivatives, prepared with the aim of obtaining new drugs for photochemotherapy, has been studied. The more interesting compounds are 4-hydroxymethyl-4',5'-benzopsoralen and 4-hydroxymethyl-4',5'-tetrahydro-benzopsoralen, which were found to be active in the dark also: DNA and RNA synthesis were both inhibited in Ehrlich cells, even if in a partially reversible fashion, while protein synthesis remained unaffected. In Chinese hamster ovary cells cultured in vitro, the clonal growth was strongly inhibited by incubation in the dark with both drugs, while a number of chromosomal aberrations was observed in the fraction of growing cells. Using alkaline elution, DNA strand breaks were detected. In addition, in the presence of aphidicolin, a specific inhibitor of DNA polymerase, the clonal growing capacity was completely restored; in contrast, the number of DNA strand breaks remained unchanged. All these results suggest that DNA topoisomerases are probably the target of these two benzopsoralens. These compounds are also good sensitizers; by UV-A irradiation they have a good capacity to produce singlet oxygen, but they appeared to be unable to induce erythemas on guinea-pig skin. Under UV-A light, they induced a strong inhibition of DNA synthesis in Ehrlich cells. Thus, benzopsoralens appear to be capable of inducing strong antiproliferative effects by two different mechanisms, by UV-A irradiation and in the dark.

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.

Trivalent chromium is neither cytotoxic nor mutagenic in permeabilized hamster fibroblasts.

BHK cells became reversibly permeable by a 30-min incubation in hypertonic medium. During permeabilization they were exposed to water-soluble Cr(VI) (K2Cr2O7) and Cr(III) (CrCl3). Thymidine uptake in the intracellular nucleotide pool, DNA replication, DNA damage and repair and sister-chromatid exchanges (SCE) were examined to detect the cytotoxic and genetic effects of Cr compounds. Cr(III) remained inactive also in permeabilized cells. An apparent induction of DNA damage by Cr(III), suggested by the Painter's test, was considered unreliable. Cr(VI) cytotoxic and genetic activity was enhanced in permeabilized cells, as demonstrated by increased inhibition of DNA replication and higher frequency of SCE.

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.

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 chromosomal lesions induced in mouse bone marrow cells by mitomycin C, as evaluated by SCE analysis.

The frequency of sister-chromatid exchanges (SCE) was evaluated in mouse bone marrow cells at different time intervals (from 19 h to 10 days) after treatment i.p. with mitomycin C (MMC; 1 and 2 mg/kg body weight). Significantly higher frequencies of SCE were found during the first week after treatment, at both doses tested. This result confirms that chromosomal lesions induced by MMC in the mouse may persist in bone marrow cells, in agreement with previous evidence based on chromosomal aberration analysis in the same cell population. In addition, the observation of a unimodal distribution of SCE/cell frequencies at each time tested indicates that the bone marrow cell population on the whole is affected by increased SCE frequency, i.e., that persistent chromosomal lesions may be transmitted along with cell proliferation.

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.

Persistence of chromosomal lesions induced in actively proliferating bone marrow cells of the mouse.

The persistence of chromosomal lesions induced in vivo by mitomycin C (MMC) was evaluated by cytogenetic analysis of mouse bone marrow cells. Chromosome aberration (CA) and micronucleus (MN) frequencies were estimated at different times after treatment, up to 42 days. The frequency of CA per cell decreased in the first 3 days after treatment, but a secondary peak appeared on the 4th day, followed by a stabilization around 0.03 CA per cell (significantly different from the control value), which persisted up to 17 days. At the next time intervals tested (28 and 42 days), the CA frequency returned to the control level. In disagreement with these data obtained directly on metaphases, the MN frequency, as evaluated in polychromatic erythrocytes, decreased quickly after treatment, reaching the control value on the 5th day. We attempted to enhance the sensitivity of the MN test by using CREST antibodies and indirect immunofluorescence. However, higher proportions of CREST- MN in treated than in control animals were observed only at short time intervals, confirming the results obtained with the conventional MN assay.

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.

Relationship between DNA replicon size and SCE induction in BALB/c and BALB/Mo mouse lymphocytes.

We studied the DNA replicon size in BALB/c and BALB/Mo mouse lymphocytes by the method of bromodeoxyuridine photolysis. After treatment of the BALB/Mo lymphocytes in vitro with mitomycin C, the average DNA replicon size appeared to be significantly smaller than that observed in BALB/c lymphocytes treated similarly. In these conditions an increased susceptibility to SCE induction in BALB/Mo lymphocytes had been observed. In the presence of both mitomycin C and cordycepin (an antiviral drug), both the DNA replicon size and the SCE frequency returned to normal values.

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.

Clastogenic effect of the human T-cell leukemia virus type I Tax oncoprotein correlates with unstabilized DNA breaks.

Expression of the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein rapidly engenders DNA damage as reflected in a significant increase of micronuclei (MN) in cells. To understand better this phenomenon, we have investigated the DNA content of MN induced by Tax. Using an approach that we termed FISHI, fluorescent in situ hybridization and incorporation, we attempted to characterize MN with centric or acentric DNA fragments for the presence or absence of free 3'-OH ends. Free 3'-OH ends were defined as those ends accessible to in situ addition of digoxigenin-dUTP using terminal deoxynucleotidyl transferase. MN were also assessed for centromeric sequences using standard fluorescent in situ hybridization (FISH). Combining these results, we determined that Tax oncoprotein increased the frequency of MN containing centric DNA with free 3'-OH and decreased the frequency of MN containing DNA fragments that had incorporation-inaccessible 3'-ends. Recently, it has been suggested that intracellular DNA breaks without detectable 3'-OH ends are stabilized by the protective addition of telomeric caps, while breaks with freely detectable 3'-OH are uncapped and are labile to degradation, incomplete replication, and loss during cell division. Accordingly, based on increased detection of free 3'-OH-containing DNA fragments, we concluded that HTLV-I Tax interferes with protective cellular mechanism(s) used normally for stabilizing DNA breaks.

Identification of kinetochore-containing (CREST+) micronuclei in mouse bone marrow erythrocytes.

A methodology for the characterization of kinetochore-containing (CREST+) micronuclei (MN), based on the use of antikinetochore antibodies (derived from CREST patients) and indirect immunofluorescence, was applied to mouse bone marrow erythrocytes. The proposed protocol allows us to obtain fluorescent signals of good quality and highly reproducible data. The clastogenic agent mitomycin C (MMC; 1 mg/kg body wt) and the two aneugenic compounds chloral hydrate (CH; 200 mg/kg body wt) and colchicine (COL; 1 mg/kg body wt) were used to verify the sensitivity of this approach to chemicals with different mechanisms of action. These compounds were tested at a 20 h time interval from treatment and all of them were able to significantly increase (P less than 0.001) the frequency of MN in polychromatic erythrocytes. Of the MN observed in preparations from control animals, 45% were CREST+ and this percentage increased significantly (P less than 0.001) after treatment with CH or COL. On the contrary, only 22% CREST+ MN were found after treatment with MMC (statistical comparison with the control value: P less than 0.001). The CREST characterization of MN induced in vivo in mouse bone marrow allows us to infer the origin of MN formation, thus contributing to the identification of aneugenic agents.

Cytotoxic and clastogenic effects of soluble chromium compounds on mammalian cell cultures.

The inhibition of cell growth, the reduction of cell survival and the induction of chromosome aberrations and of sister chromatid exchange (SCE) have been determined in cultured hamster cell lines (BHK and CHO) treated with 11 water-soluble compounds of hexavalent and trivalent chromium. All Cr6+ compounds inhibit growth of BHK cells and reduce survival of CHO cells to levels comparable to those obtained only after exposure to 100--1000 times higher Cr3+ concentrations. The cytotoxicity curves obtained with the different Cr6+ compounds are almost overlapping, whereas marked differences of activity are noticeable among Cr3+ compounds. Giant cells are obtained after exposure to Cr6+ and Cr3+ compounds, as shown by the rise of DNA and RNA per cell, and are due to the blockage of the cell cycle without sudden inhibition of macromolecular syntheses. Both Cr6+ and Cr3+ compounds are able to induce chromosome aberrations, whereas Cr3+ is absolutely incapable of inducing SCE, only Cr6+ being active. The frequency of chromosome aberrations is increased about 10-fold after exposure to 1.0 micrograms/ml Cr6+, whereas it is only doubled after treatment with up to 150 micrograms/ml Cr3+. On the other hand, in spite of the sensitivity of CHO cells to the induction of SCE by mitomycin C, the frequency of SCE hardly doubles after exposure to Cr6+ compounds. The present data confirm that Cr6+ compounds are characterized by a marked cytotoxicity and clastogenic action on mammalian cell cultures and show that Cr3+ compounds, though cytotoxic only at extremely high concentrations and not increasing the frequency of SCE, are not completely without cytogenetic effect, as they are able to induce chromosome aberrations.

Cytotoxic and clastogenic effects of soluble and insoluble compounds containing hexavalent and trivalent chromium.

Cr(III) and Cr(VI) compounds of varying solubilities have been tested in vitro for their ability to inhibit cell growth and nucleic acid and protein syntheses in BHK cells, to induce alterations in the mitotic cycle in HEp cells, and to increase the frequency of chromosomal aberrations and sister chromatid exchanges (SCE) in CHO cells. All Cr(VI) compounds, and particularly those containing soluble Cr(VI), such as potassium dichromate and zinc yellow, differentially inhibit macromolecular syntheses in BKH cells, that of DNA being always the most affected. Among Cr(III) compounds, which generally have very low cytotoxicity, chromite is particularly active, and inhibits cell growth and DNA synthesis even more than the poorly soluble Cr(VI) compounds. Preincubation in growth medium, with or without metabolizing cell cultures, solubilizes considerable amounts of Cr(VI) from zinc yellow and chromite, but significant amounts are also obtained from the most insoluble Cr(VI) pigments. When BHK cells are treated with such preincubated solutions, reduction of soluble Cr(VI) to Cr(III) by cell metabolites is seen with all Cr(VI) compounds, accompanied by decreased cytotoxicity. The same differences between Cr(VI) and Cr(III) compounds apply to the cytotoxic effects on mitosis of HEp cells and the clastogenic effects on CHO cells. The activity of chromite, the only Cr(III) pigment capable of significantly increasing the frequency of SCE, is due to contamination with soluble Cr(VI). In contrast to the very low cytotoxicity of Cr(III), much higher chromium levels are detected in the cells incubated with soluble Cr(III) than with the same concentrations of soluble Cr(VI). 50% and 75% of chromium accumulated in the cells during treatments with Cr(VI) and Cr(III) respectively remains firmly bound to the cells, even when they are incubated for up to 48 h in normal growth medium. Chromium accumulated in the cells after treatment with Cr(III) is most probably bound to the cell membrane, whereas some of the Cr(VI) is transported through the cell membrane and reduced in the cell nucleus. The results of the present investigation are in agreement with those obtained with the same Cr(VI) and Cr(III) compounds in mutagenicity assays in bacteria and carcinogenicity tests in rodents. A re-evaluation of the mechanisms of chromium carcinogenisis is proposed.