Phytohemagglutinin-stimulated Human Lymphocytes Research Articles

Antiproliferative effects of selective adenosine receptor agonists and antagonists on human lymphocytes: evidence for receptor-independent mechanisms.

The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A1, A2A, and A2B ARs. Cell proliferation was measured by [(3)H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A1), BAY60-6583 (A2B), and IB-MECA (A3), and the antagonists PSB-36 (A1), MSX-2 (A2A), and PSB-10 (A3) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A2A), and the antagonists preladenant (SCH-420814, A2A), PSB-1115 (A2B), and PSB-603 (A2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC50 value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.

Immunomodulatory effect of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells on lymphocytes

Studies have shown that mesenchymal stem cells (MSCs) have low immunogenicity and immune regulation. Human umbilical cord Wharton’s jelly provides a new source for MSCs that are highly proliferative and have multi-differentiation potential. To investigate immunomodulatory effects of human Wharton’s jelly cells (WJCs) on lymphocytes, we successfully isolated MSCs from human umbilical cord Wharton’s jelly. WJCs expressed MSC markers but low levels of human leukocyte antigen (HLA)-ABC and no HLA-DR. These results indicate that WJCs have low immunogenicity. Both WJCs and their culture supernatant could inhibit the proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes and mouse splenocytes. Additionally, WJCs suppressed secretion of transforming growth factor-β1 and interferon-γ by human peripheral blood lymphocytes. We conclude that the immunomodulatory effect of WJCs may be related to direct cell contact and inhibition of cytokine secretion by human peripheral blood lymphocytes.

Titanium dioxide nanoparticles induce apoptosis through the JNK/p38-caspase-8-Bid pathway in phytohemagglutinin-stimulated human lymphocytes

We investigated the signaling pathways underlying nano-TiO2-induced apoptosis in cultured human lymphocytes. Nano-TiO2 increased the proportion of sub-G1 cells, activated caspase-9 and caspase-3, and induced caspase-3-mediated PARP cleavage. Nano-TiO2 also induced loss of mitochondrial membrane potential, which suggests that nano-TiO2 induces apoptosis via a mitochondrial pathway. A time-sequence analysis of the induction of apoptosis by nano-TiO2 revealed that nano-TiO2 triggered apoptosis through caspase-8/Bid activation. We also observed that inhibition of caspase-8 by z-IETD-fmk suppressed the caspase-8/Bid activation, caspase-3-mediated PARP cleavage, and apoptosis. Nano-TiO2 activated two MAPKs, p38 and JNK. In addition, the selective p38 inhibitor SB203580 and selective JNK inhibitor SP600125 suppressed nano-TiO2-induced apoptosis and caspase-8 activation to moderate and significant extents, respectively. Knockdown of protein levels of JNK1 and p38 using an RNA interference technique also suppressed caspase-8 activation. Our results suggest that nano-TiO2-induced apoptosis is mediated by the p38/JNK pathway and the caspase-8-dependent Bid pathway in human lymphocytes.

Lymphocyte proliferation stimulated by activated human macrophages treated with Canova

Canova (CA) is a homeopathic medication with immunomodulatory properties, recommended for patients with a depressed immune system. CA has been reported to increase in leukocyte numbers, cellular differentiation and reduction in tumor size. Since CA may stimulate lymphocyte differentiation, proliferation, and/or survival, the aim of the present study was to compare the mitotic index (MI) of phytohemagglutinin-stimulated human lymphocytes cultured in a medium supplemented with human macrophages activated by CA, with lymphocytes cultured in a medium without CA-treated macrophages. In this study, the MI of lymphocyte cultured received the medium containing CA-stimulated macrophages showed a higher proliferation index (p<0.01) than the lymphocytes cultured in a medium without CA-treated macrophages. Our results suggest that CA treatment, in addition to activating macrophages, indirectly induces lymphocyte proliferation and has potential as a new adjuvant therapeutic approach.

Evidence of melatonin synthesis by human lymphocytes and its physiological significance: possible role as intracrine, autocrine, and/or paracrine substance.

It has been historically assumed that the pineal gland is the major source of melatonin (N-acetyl-5-methoxytryptamine) in vertebrates. Melatonin plays a central role in fine-tuning circadian rhythms in vertebrate physiology. In addition, melatonin shows a remarkable functional versatility exhibiting antioxidant, oncostatic, antiaging, and immunomodulatory properties. Melatonin has been identified in a wide range of organisms from bacteria to human beings. Its biosynthesis from tryptophan involves four well-defined intracellular steps catalyzed by tryptophan hydroxylase, aromatic amino acid decarboxylase, serotonin-N-acetyltransferase, and hydroxyindole-O-methyltransferase. Here, for the first time, we document that both resting and phytohemagglutinin-stimulated human lymphocytes synthesize and release large amounts of melatonin, with the melatonin concentration in the medium increasing up to five times the nocturnal physiological levels in human serum. Moreover, we show that the necessary machinery to synthesize melatonin is present in human lymphocytes. Furthermore, melatonin released to the culture medium is synthesized in the cells, because blocking the enzymes required for its biosynthesis or inhibiting protein synthesis in general produced a significant reduction in melatonin release. Moreover, this inhibition caused a decrease in IL-2 production, which was restored by adding exogenous melatonin. These findings indicate that in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that this melatonin could be involved in the regulation of the human immune system, possibly by acting as an intracrine, autocrine, and/or paracrine substance.

Cyclin D3 and p53 mediate sulforaphane-induced cell cycle delay and apoptosis in non-transformed human T lymphocytes.

Despite experimental evidence that sulforaphane can exert chemopreventive effects, whether these effects are specific for neoplastic cells is not known. Following our previous demonstration that sulforaphane induces cell cycle arrest and apoptosis in human T lymphoblastoid Jurkat leukemia cells and increases p53 and bax protein expression, we tested sulforaphane on non-transformed phytohemagglutinin-stimulated human lymphocytes. Here, we demonstrate that sulforaphane arrested cell cycle progression in G, phase, through a decrease in the protein expression of cyclin D3. Moreover, sulforaphane induced apoptosis (and also necrosis), mediated by an increase in the expression of p53. These findings suggest that sulforaphane is a growth modulator for T cells. Our in vitro evidence that sulforaphane is active and even cytotoxic in normal as well as transformed lymphocytes raises important questions regarding its suitability for cancer chemoprevention.

Factors influencing the removal of thymine glycol from DNA in γ-irradiated human cells

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.

Cisplatin at clinically relevant concentrations enhances interleukin-2 synthesis by human primary blood lymphocytes.

Cytotoxic drugs influence the expression of certain genes in cancer cells. Cisplatin has recently been shown to modulate interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha production in macrophages. In this study, we wanted to investigate whether cisplatin interferes with the IL-2, IL-2 receptor (IL-2R), interferon (IFN)-gamma, and TNF-alpha expression in phytohemagglutinin-stimulated human peripheral blood lymphocytes. IL-2 was analyzed in a bioassay, while IFN-gamma and TNF-alpha were measured by ELISA. Northern blots were performed to quantify steady-state cytokine mRNA levels. Furthermore, T cell subsets and IL-2R surface expression were analyzed by means of flow cytometry. A maximum stimulatory effect on IL-2 production (1.8-fold increase) was observed with cisplatin at 5-10 microM while IFN-gamma and TNF-alpha synthesis and IL-2R density were unaffected. However, cisplatin-treated cells displayed enhanced IL-2, IL-2R, IFN-gamma and TNF-alpha mRNA levels compared to drug-free controls. Cisplatin did not prolong cytokine mRNA half-life as revealed with the transcriptional inhibitor actinomycin D. In contrast to an inhibited growth of CD4+ T lymphocytes, CD3+ CD8+ cell density was unaffected at intermediate cisplatin concentrations (10 microM). Bleomycin, carboplatin, doxorubicin, novobiocin or etoposide, which were included for comparison, did not interfere with IL-2 expression. Our data imply that cisplatin most likely stimulated cytokine transcription via a putative stress-induced signaling pathway.

In vitro modulation of activation antigens on human lymphocytes by beta-estradiol.

The possible in vitro immunomodulating effect of beta-estradiol on phytohemagglutinin-stimulated human lymphocyte cultures was studied. Lymphocyte cultures from 12 healthy men and women aged 25-35 years were set up for 12 hr in the presence and in the absence of beta-estradiol, and the expression of the activation markers CD25, CD69, and CD71 was examined by flow cytometric analysis with specific fluorescent conjugated antibodies. Although the number of cases is small, in 10 of 12 cases in the presence of beta-estradiol in two different concentrations, a significantly decreased expression of CD69 could be observed. A slight decrease could also be observed for the Interleukin-2 receptor expression; however, the difference, in the presence or absence of beta-estradiol, was not significant. The results suggest that in vitro addition of beta-estradiol can inhibit, to a certain degree, specific activation markers on phytohemagglutinin-stimulated lymphocytes from young men and women. The present study could not define the role of sex differences because of the small number of samples. A comparison between men and women at various ages in a greater number of cases, as well as studies on activation markers after treatments with estrogens, would be useful.

Activation markers and cell proliferation as indicators of toxicity: a flow cytometric approach.

Cell proliferation is an attractive endpoint in in vitro toxicity assays, since nearly any kind of damage in a cell may result in altered cell proliferation. In toxicological applications, liquid scintillation counting, measuring radioactivity from tritiated thymidine, has been the traditional way to estimate cell proliferation. An alternative approach is the measurement of BrdU incorporation by flow cytometry. Before the actual DNA synthesis starts, several proteins are expressed on the cell surface, as well as intracellularly. Among the markers on the cell surface CD69, CD25, and CD71 are sequentially expressed on human lymphocytes after a mitogenic stimulation. The aim of this study was to evaluate information obtained by analysis of expression of activation markers on cell surfaces in lymphocyte subsets and to compare it with data from cell proliferation studies performed by liquid scintillation counting and BrdU flow cytometry. The experiments were performed with phytohemagglutinin-stimulated human lymphocytes exposed to ochratoxin A and cyclosporin A. While ochratoxin A-treated cultures showed a steep inhibition with increasing concentration, the cyclosporin A treatment gave an inhibition curve with a less steep slope. Activation marker studies showed that the effect of treatment with both of the toxins was more pronounced on the late markers CD25 and CD71, while CD69 had the advantage that significant effects could be detected as early as 6 h after ochratoxin A treatment. Cyclosporin A treatment induced only minor alterations in CD69 expression. Certain differences in expression of activation markers between CD4+ and CD8+ subsets were found both in ochratoxin A- and cyclosporin A-treated cultures. A stimulating effect was found in cell cultures exposed to the lowest concentration of ochratoxin A on CD69 and CD25 expression. Signs of an increase in frequencies of proliferating cells measured with the BrdU flow cytometry method were also seen. This increase could not be detected with liquid scintillation counting. No other differences between the liquid scintillation counting and BrdU flow cytometry measurements of proliferation were obtained. We conclude that studies of activation marker expression by the flow cytometric approach used in this report are useful complements to traditional measurements of cell proliferation as they yield subset-specific information about cellular processes which precede proliferation of lymphocytes.

An Ion-Pairing High-Performance Liquid Chromatographic Method for the Direct Simultaneous Determination of Nucleotides, Deoxynucleotides, Nicotinic Coenzymes, Oxypurines, Nucleosides, and Bases in Perchloric Acid Cell Extracts

An ion-pairing high-performance liquid chromatographic method for the direct and simultaneous determination of nucleotides, deoxynucleotides, cAMP, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts is presented. By using an Alltima C-18, 250 × 4.6-mm, 5-μm particle size column, a high resolution of 38 acid-soluble compounds, including ATP, GTP, dTTP, CTP, UTP, ADP, GDP, dTDP, CDP, UDP, dATP, dGTP, dCTP, dUTP, dADP, dGDP, dCDP, dUDP, and cAMP, is obtained. Elution is performed with a step gradient from buffer A (consisting of 10 mMtetrabutylammonium hydroxide, 10 mMKH2PO4, 0.25% methanol, pH 7.00) to buffer B (consisting of 2.8 mMtetrabutylammonium hydroxide, 100 mMKH2PO4, 30% methanol, pH 5.50). Perchloric acid extracts of resting and phytohemagglutinin-stimulated human lymphocytes were analyzed. Data indicate that this chromatographic method offers, for the first time to the best of our knowledge, the possibility of simultaneously determining di- and triphosphate nucleosides and their corresponding deoxynucleosides without any chemical manipulation of samples except for perchloric acid deproteinization. Hence, the present HPLC assay minimizes the risks of modification or loss of metabolite concentration and allows one to obtain, with a single chromatographic run, the complete pattern of those metabolites which are known to be involved in energy metabolism and in DNA and RNA synthesis, resulting therefore of great advantage in cell biology studies.

Human thymidine kinase 1. Regulation in normal and malignant cells

In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 °C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with K m values of 14 μ m and 0.5 μ m, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3–5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G 0/G 1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with K m values of 0.3–0.4 μ m regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the K m values of TK1 from these cells were 45 μ m for the ATP-free enzyme and 65 μ m for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high K m values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.

Assignment of the DPP4 Gene Encoding Adenosine Deaminase Binding Protein (CD26/Dipeptidylpeptidase IV) to 2q23

FISH was performed on chromosome preparations obtained from phytohemagglutinin-stimulated human blood lymphocytes. cDNA encoding ADAbp was isolated from the SK-RC-28 human renal cell carcinomas cell line using PCR technique and was cloned in pSVK3 plasmid for use as a probe. The PCR primers were constructed from the known nucleotide sequence of CD26, and the cDNA product was extracted from nucleotides 1 to 2344. The vector containing the probe was labeled by nick-translation with biotin-11-dUTP. Hybridization to chromosome spreads, washings, detection with FITC-conjugated avidin, selection and photography of metaphases, analysis of signals, and banding were performed according to the described method.

Application of laser scanning image cytometry for human chromosome microdissection and DNA analysis

P. METEZEAU, G. TACHDJIAN*, H. KIEFER, L. DRUART, V. CACHEUX*, C. NESSMANN* and G. LIZARD Biochimie Cellulaire et Cytometrie Analytique et Preparative, SC 9 INSERM-Pasteur, 28 rue du DrRoux, 75724 Paris cedex 15, France * Unite de Cytogenetique, Service de Biologie du Developpement et de la Reproduction, Hopital Robert Debre, 48 Boulevard Serurier, 75019 Paris, France Centre Commun Cytometrie en Flux, HopitalE. Herriot, Place d'Arsonval, 69437Lyon cedex 03, France INTRODUCTION Recent progress in molecular biology have increased the use of chromosomal microdissection in human genetics (Kao and Yu, 1991; Seki et al., 1993). Indeed, generation of DNA probes from genome is of great interest in Genetics. This objective can be obtained by using microdissection and amplification of a desired chromosomal region (Ludecke et al., 1989; Meltzer et al., 1992). We describe here application of laser scanning image cytometry for human chromosome microdissection and DNA analysis. MATERIALS AND METHODS Chromosome preparation Phytohemagglutinin-stimulated human lymphocyte cultures were established from healthy donors. Metaphase spreads were prepared according to our standard protocoles. Laser microdissection The laser microdissection of the chromosomes was performed on chromosomes stained with Giemsa 0.5%. Conditions adopted for current microdissection were obtained with

Increased poly(ADP-ribose) polymerase activity during repair of (+/-)-anti-benzo[a]pyrene diolepoxide-induced DNA damage in human peripheral blood lymphocytes in vitro.

Poly(ADP-ribose) polymerase, which catalyzes the formation of poly(ADP-ribose) polymers, is an enzyme involved in cell proliferation, differentiation and transformation as well as in recovery from DNA damage. Poly(ADP-ribose) polymers are rapidly synthesized from the ADP-ribose moieties from intracellular NAD+, which, as a consequence, is depleted. It has been shown that DNA strand breaks are required for enzyme activation and it is suggested that one of the functions of poly(ADP-ribosylation) is to improve accessibility of damaged sites to other DNA repair enzymes. The aim of this study was to investigate whether poly(ADP-ribosylation) is involved in repair of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE]-induced DNA damage in human lymphocytes in vitro. Results show that (+/-)-anti-BPDE is capable of inducing poly(ADP-ribosylation), NAD+ depletion and inhibition of proliferation in phytohemagglutinin-stimulated human peripheral blood lymphocytes. Also, repair of (+/-)-anti-BPDE induced DNA damage was confirmed by both unscheduled DNA synthesis and (+/-)-anti-BPDE-deoxyguanosine adduct removal. Based on these findings, it is concluded that poly(ADP-ribosylation) is involved in (+/-)-anti-BPDE-induced DNA repair in these cells. In addition, these results confirm the possible relation between poly(ADP-ribosylation), NAD+ depletion and inhibition of proliferation, after induction of DNA damage.

P145 expression during the cell cycle in HL-60 cell line and normal human lymphocytes: Effects of camptothecin, vinblastine, cycloheximide, actinomycin D, retinoic acid and DMSO

Bivariate flow cytometric analysis of nucleolar antigen p145 was performed on quiescent and phytohemagglutinin-stimulated human lymphocytes and on a promyelocytic cell line (HL-60). Data were acquired on a FACScan flow cytometer and analyzed using LYSYS II. Quiescent lymphocytes did not express p145. PHA-stimulated proliferating lymphocytes expressed p145 maximally after 48 h, similarly to HL-60 cells in exponential growth. Antigen expression in G 1 was notably heterogeneous in both cell types. The ratio of p145/DNA was highest in early S and decreased during mid and late S and remained low in G 2M. p145 expression was lowest in M-phase cells treated for 6 h with vinblastine. Cycloheximide and actinomycin D had similar effects on p145 in HL-60 cells: expression of p145 gradually decreased from 1 to 6 h incubation in all phases of the cell cycle. Camptothecin did not decrease p145 expression and apoptotic cells from CAM-treated cultures still expressed p145. Retinoic acid and DMSO induced differentiation in HL-60 cells, and as this process progressed, p145 levels gradually fell until they approached isotype antibody control levels at 9 and 6 days, respectively. However, after 5 days treatment with 2 nM retinoic acid apoptotic cells appeared which still expressed p145. The data on drug treatment suggest that p145 exists in undifferentiated and proliferating cells and may not be a specific marker for malignancy, but may prove useful as a monitor of chemotherapeutic effects in cancer treatment.

Effects of progesterone and estradiol on the proliferation of phytohemagglutinin-stimulated human lymphocytes

In this paper we report on a study to elucidate whether the response of human lymphocytes to mitogenic stimulation was modified by physiological changes which occur during the menstrual cycle. Experiments with untreated cultures showed intra-individual variation to mitogen stimulation in female lymphocyte cultures, but a significant correlation between the menstrual cycle and the proliferation kinetics of lymphocytes was not found. Consequently, we performed experiments in which two of the hormones that regulate the menstrual cycle in women, estradiol and progesterone, were added to cultured human lymphocytes obtained from both men and women. The results indicate that both hormones at physiological concentrations have the capacity to modify the proliferation of PHA-stimulated human lymphocytes. Therefore, both hormones could play a role in the induction of the intra-individual variation observed in the untreated female cultures. However, in vivo other factors could also modify the proliferation kinetics of human lymphocytes preventing the demonstration of the effects of a single factor, such as the hormonal changes occurring during the menstrual cycle.

Mechanisms of AFF-induced slippage in contiguous sequences: evidence that AAF adducts can induce both targeted and semitargeted -1 frameshift mutations

An ion-pairing high-performance liquid chromatographic method for the direct and simultaneous determination of nucleotides, deoxynucleotides, cAMP, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts is presented. By using an Alltima C-18, 250 × 4.6-mm, 5-μm particle size column, a high resolution of 38 acid-soluble compounds, including ATP, GTP, dTTP, CTP, UTP, ADP, GDP, dTDP, CDP, UDP, dATP, dGTP, dCTP, dUTP, dADP, dGDP, dCDP, dUDP, and cAMP, is obtained. Elution is performed with a step gradient from buffer A (consisting of 10 mMtetrabutylammonium hydroxide, 10 mMKH2PO4, 0.25% methanol, pH 7.00) to buffer B (consisting of 2.8 mMtetrabutylammonium hydroxide, 100 mMKH2PO4, 30% methanol, pH 5.50). Perchloric acid extracts of resting and phytohemagglutinin-stimulated human lymphocytes were analyzed. Data indicate that this chromatographic method offers, for the first time to the best of our knowledge, the possibility of simultaneously determining di- and triphosphate nucleosides and their corresponding deoxynucleosides without any chemical manipulation of samples except for perchloric acid deproteinization. Hence, the present HPLC assay minimizes the risks of modification or loss of metabolite concentration and allows one to obtain, with a single chromatographic run, the complete pattern of those metabolites which are known to be involved in energy metabolism and in DNA and RNA synthesis, resulting therefore of great advantage in cell biology studies.

Persistent inapparent HIV-1 infection of human neuroblastoma cells

We have studied human immunodeficiency virus type 1 (HIV-1) infection in three different human neuroblastoma cell lines; SK-N-MC, IMR-32 and SH-SY5Y. In all of these cell lines the infection became productive. However, the virus expression was different as determined by the p24 antigen capture assays from culture supernatants and immunochemical (APAAP) staining of cells. The medium of SK-N-MC cells contained approximately 300 pg p24 antigen per 10(6) cells, 0.1-1% of the cells were p24 antigen-positive and characteristic genomic and subgenomic HIV mRNA species were seen in Northern blotting. In infected IMR-32 and SH-SY5Y cell cultures, the HIV-1 production was below the level of detection. However, infectious virus was found by inoculating cultures of the lymphoid cell C8166 with the cell-free supernatant fluid from the neuroblastoma cultures. The lymphoid cells became positive within one week. Moreover, phytohemagglutinin-stimulated normal human lymphocytes produced virus, if cocultured with any of the three infected neuroblastoma cell lines. The infection was persistent and has been followed, using the above techniques, for 4 months in the case of SK-N-MC and IMR-32 cells and 6 months in the case of SH-SY5Y cells. During this period, no alterations in cell morphology, viability, or proliferative capacity were seen. All three neuroblastoma lines were negative for the CD4 receptor mRNA according to Northern hybridization and RNase protection assays. We conclude that HIV-1 produces persistent and inapparent infection in human neuroblastoma cells, using a CD 4-independent mechanism of entry to the cells.

Spontaneous and mitomycin-C-induced micronuclei in human lymphocytes exposed to extremely low frequency pulsed magnetic fields

The cytokinesis block micronucleus method, a very sensitive cytogenetic assay, was used to ascertain the possible genotoxic effects of extremely low frequency pulsed magnetic fields in phytohemagglutinin-stimulated human lymphocytes cultures from 16 healthy donors. Four conditions were studied: i) lymphocytes not exposed to the field (control cultures); ii) lymphocytes exposed to the field; iii) lymphocytes treated with mitomycin-C and not exposed to the field; iv) lymphocytes treated with mitomycin-C and exposed to the field. Mitomycin-C-treated cultures were used as control for the micronucleus method, because it is known that mitomycin-C is a potent genotoxic agent, capable of inducing micronuclei. The frequency of micronuclei in field-exposed cultures was similar to the spontaneous frequency observed in control unexposed-cultures. Moreover, the exposure to pulsed magnetic fields did not affect the frequency of micronuclei induced by mitomycin-C, suggesting that, in the experimental conditions used, this kind of field neither affected the integrity of chromosomes nor interfered with the genotoxic activity of mitomycin-C.