Proliferation Of Human Cell Lines Research Articles

3,4,5-Trimethoxychalcones Tubulin Inhibitors with a Stable Colchicine Binding Site as Potential Anticancer Agents

The development of microtubule perturbing drugs is one of the most promising anticancer therapeutic methods. Unfortunately, limitation such as drug resistance, adverse side effects, complex formulations and synthesis, and limited bioavailability of these microtubule perturbing drugs has aroused the search for a new molecule of the tubulin system. Different substituents of chalcone were designed, synthesized, and determined for inhibition of tubulin assembly and toxicity in human cancer cell lines based on conventional colchicine site ligands and a computer model of the colchicine binding site on tubulin. A molecular docking study indicated that the chalcone scaffold could fit the colchicine site on tubulin in a similar orientation to the natural product. The 3,4,5-trimethoxyphenyl ring, which occupies the same sub-cavity as the equivalent molecule in colchicine, appeared to benefit the ligand of α,β-tubulin interaction. Several 3,4,5-trimethoxychalcone compounds demonstrated improved cytotoxicity against MCF-7 cells and inhibited tubulin assembly in vitro as potently as colchicine. The most active chalcone 1 with the IC50 of 6.18 ± 0.69 μM prevented the proliferation of human cell lines at micromolar concentrations, causing microtubule destabilization and mitotic arrest in humans inhibiting breast cancer cells.

The antimalarial drug amodiaquine stabilizes p53 through ribosome biogenesis stress, independently of its autophagy-inhibitory activity

Pharmacological inhibition of ribosome biogenesis is a promising avenue for cancer therapy. Herein, we report a novel activity of the FDA-approved antimalarial drug amodiaquine which inhibits rRNA transcription, a rate-limiting step for ribosome biogenesis, in a dose-dependent manner. Amodiaquine triggers degradation of the catalytic subunit of RNA polymerase I (Pol I), with ensuing RPL5/RPL11-dependent stabilization of p53. Pol I shutdown occurs in the absence of DNA damage and without the subsequent ATM-dependent inhibition of rRNA transcription. RNAseq analysis revealed mechanistic similarities of amodiaquine with BMH-21, the first-in-class Pol I inhibitor, and with chloroquine, the antimalarial analog of amodiaquine, with well-established autophagy-inhibitory activity. Interestingly, autophagy inhibition caused by amodiaquine is not involved in the inhibition of rRNA transcription, suggesting two independent anticancer mechanisms. In vitro, amodiaquine is more efficient than chloroquine in restraining the proliferation of human cell lines derived from colorectal carcinomas, a cancer type with predicted susceptibility to ribosome biogenesis stress. Taken together, our data reveal an unsuspected activity of a drug approved and used in the clinics for over 30 years, and provide rationale for repurposing amodiaquine in cancer therapy.

Abstract 3295: CAGI: The Critical Assessment of Genome Interpretation, a community experiment to evaluate phenotype prediction: implications for predicting impact of variants in cancer

Abstract Interpretation of genetic variants plays an essential role in cancer and other diseases. Needs for variant interpretation span from basic research to informing profound clinical decisions. The Critical Assessment of Genome Interpretation (CAGI, \'kā-jē\) is a community experiment to objectively assess computational methods for predicting the phenotypic impacts of genomic variation. CAGI participants are provided genetic variants and make predictions of resulting phenotype. These predictions are evaluated against experimental or clinical characterizations by independent assessors. CAGI establishes objective reference standards on how well current computational methods do—and do not—meet clinical and research requirements. As such CAGI enables appropriate use of methods and promotes the development of improved approaches. There have been notable discoveries from each of the four CAGI experiments to date, and general themes have emerged. Some examples illustrating the themes: a challenge, in which participants had to blindly predict how missense variants in p16 affect proliferation in human cell lines, showed that individual variants prediction for even the top performing methods were not consistently accurate. The RAD50 challenge in which participants were asked to classify RAD50 variants in breast cancer cases and controls, along with other challenges, showed that missense methods tend to correlate better with each other than with experiment (for reasons that may reflect biases in the predictive methods but also in the experimental assays). Bespoke approaches often enhance performance, as seen for example in also the RAD50 challenge, where the use of the knowledge of which domains are involved in DNA repair resulted in more accurate performance. Results from a challenge in which participants had to predict whether p53 core domain mutations recue activity of inactive p53 showed that protein three-dimensional structure-based missense methods do well in a few cases, while sequence-based methods have more consistent performance on most challenges. A challenge in which predictors predicted the response of 54 breast cancer cell lines to a panel of cancer drugs showed that training the model with external data enabled to find the correct ballpark of drug sensitivity. Interpretation of non-coding variants shows promise but is not at the level of missense; one challenge showing this had predict the probability that variants in BRCA1 and BRCA2 collected by Free the Data are pathogenic. Two groups correctly predicted all the deletion and insertion mutations, but they were not able to correctly predict classification for all the intron mutations. CAGI findings suggest that running multiple uncalibrated methods and considering their consensus may result in undue confidence, making this procedure inadvisable. Citation Format: Gaia Andreoletti, Roger A. Hoskins, Susanna Repo, Daniel Barsky, Steven E. Brenner, John Mult, Cagi participants. CAGI: The Critical Assessment of Genome Interpretation, a community experiment to evaluate phenotype prediction: implications for predicting impact of variants in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3295.

In vitro investigation of the growth of hydroxyapatite and proliferation of human cell lines on the sol gel derived diopside co-substituted tricalcium phosphate bioceramics

Sol-gel derived bioceramic samples with the composition formula x(CaO.MgO.2SiO2).(100-x)3CaO.P2O5 where x = 0, 10, 20, 30 and 40 mass% have been prepared in the laboratory. Diopside (CaO.MgO.2SiO2) co-substituted tricalcium phosphate (3CaO.P2O5) bioceramics are speculated to be successful bone repair materials for clinical applications due to improved bone bonding ability and bioresorbability properties. Authors have employed x-ray diffraction, Raman spectroscopy, field emission scanning electron microscopy along with energy dispersive spectroscopy, hardness testing and cell culture studies during the investigations. X-ray diffraction spectra demonstrated the formation of crystalline phases of diopside, merwinite and whitlockite in the samples and Raman spectra shows the presence of phosphate species in the orthophosphate environment. In vitro hydroxyapatite forming ability in simulated body fluid has been investigated by x-ray diffraction, Raman and field emission scanning electron microscopy techniques for time durations varying between day 1 to day 12 and results demonstrated that hydroxyapatite layer has developed from day 1 in sample with composition 40 mass% tricalcium phosphate and 60 mass% diopside. Pure diopside has shown the best hardness value. Although, 10 mass% tricalcium phosphate and 90 mass% diopside composition has shown the slower rate for growth of hydroxyapatite but it has shown the best cell viability values for human cell line. Results indicate that growth rate of hydroxyapatite is composition as well as sample synthesis technique dependent.

Deletion of eIF2β lysine stretches creates a dominant negative that affects the translation and proliferation in human cell line: A tool for arresting the cell growth

ABSTRACTBackground: Eukaryote initiation factor 2 subunit β (eIF2β) plays a crucial role in regulation protein synthesis, which mediates the interaction of eIF2 with mRNA. eIF2β contains evolutionarily conserved polylysine stretches in amino-terminal region and a zinc finger motif in the carboxy-terminus.Methods: The gene eIF2β was cloned under tetracycline transcription control and the polylysine stretches were deleted by site-directed mutagenesis (eIF2βΔ3K). The plasmid was transfected into HEK 293 TetR cells. These cells were analyzed for their proliferative and translation capacities as well as cell death rate. Experiments were performed using gene reporter assays, western blotting, flow cytometry, cell sorting, cell proliferation assays and confocal immunofluorescence.Results: eIF2βΔ3K affected negatively the protein synthesis, cell proliferation and cell survival causing G2 cell cycle arrest and increased cell death, acting in a negative dominant manner against the native protein. Polylysine stretches are also essential for eIF2β translocated from the cytoplasm to the nucleus, accumulating in the nucleolus and eIF2βΔ3K did not make this translocation.Discussion: eIF2β is involved in the protein synthesis process and should act in nuclear processes as well. eIF2βΔ3K reduces cell proliferation and causes cell death. Since translation control is essential for normal cell function and survival, the development of drugs or molecules that inhibit translation has become of great interest in the scenario of proliferative disorders. In conclusion, our results suggest the dominant negative eIF2βΔ3K as a therapeutic strategy for the treatment of proliferative disorders and that eIF2β polylysine stretch domains are promising targets for this.

Pharmacodynamic and Antineoplastic Activity of BI 836845, a Fully Human IGF Ligand-Neutralizing Antibody, and Mechanistic Rationale for Combination with Rapamycin

Insulin-like growth factor (IGF) signaling is thought to play a role in the development and progression of multiple cancer types. To date, therapeutic strategies aimed at disrupting IGF signaling have largely focused on antibodies that target the IGF-I receptor (IGF-IR). Here, we describe the pharmacologic profile of BI 836845, a fully human monoclonal antibody that utilizes an alternative approach to IGF signaling inhibition by selectively neutralizing the bioactivity of IGF ligands. Biochemical analyses of BI 836845 demonstrated high affinity to human IGF-I and IGF-II, resulting in effective inhibition of IGF-induced activation of both IGF-IR and IR-A in vitro. Cross-reactivity to rodent IGFs has enabled rigorous assessment of the pharmacologic activity of BI 836845 in preclinical models. Pharmacodynamic studies in rats showed potent reduction of serum IGF bioactivity in the absence of metabolic adverse effects, leading to growth inhibition as evidenced by reduced body weight gain and tail length. Moreover, BI 836845 reduced the proliferation of human cell lines derived from different cancer types and enhanced the antitumor efficacy of rapamycin by blocking a rapamycin-induced increase in upstream signaling in vitro as well as in human tumor xenograft models in nude mice. Our data suggest that BI 836845 represents a potentially more effective and tolerable approach to the inhibition of IGF signaling compared with agents that target the IGF-I receptor directly, with potential for rational combinations with other targeted agents in clinical studies.

Low-density lipoprotein receptor-related protein 1b impairs cellular proliferation in human cell lines

Low-density lipoprotein (LDL) receptor-related protein 1b (LRP1b) is one of three very large receptors of the LDL receptor family, several members of which have been implicated in the pathogenesis of atherosclerosis. LRP1b was discovered during studies of lung cancer cell lines, where alterations of the receptor were frequently found and LRP1b was therefore postulated as a putative tumor suppressor. LRP1b is expressed in human brain, thyroid gland, skeletal muscle, and to a lesser amount in testis. Previously, we demonstrated binding of LRP1b to circulating ligands like very low density lipoprotein, high density lipoprotein, and fibrinogen. We generated an expression vector containing the full length 13.800 bp murine LRP1b cDNA for overexpression (OE) of the receptor in LRP1b-negative lung cancer cell lines (A427, A549) and used siRNA for silencing in LRP1b-positive lung cancer cells (Calu-1). Cellular proliferation was analyzed using a BrdU incorporation ELISA and anchorage-independent growth using a soft agar colony formation assay, respectively. Consistent with the postulated tumor suppressor function, overexpression of mLRP1b in A427 and A549 cells significantly reduced both cellular proliferation and anchorage-independent growth compared to mock-transfected control cells. Conversely, siRNA mediated knockdown of hLRP1b in Calu-1 lung cancer cells led to a significant increase in cellular proliferation compared to scrambled (SCR)-siRNA treated control cells (Fig 1). ![Figure][1] Figure 1 Our findings demonstrate that overexpression of full length LRP1b leads to impaired cellular proliferation, while LRP1b knockdown has the opposite effect. These experiments may help in unraveling the largely unknown physiological role of LRP1b and its potential functions in cancer and atherosclerosis. [1]: pending:yes

Doxycycline Alters Metabolism and Proliferation of Human Cell Lines

The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells–including inhibition of the mitochondrial ribosome–there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism.

Synthesis, characterization and toxicological evaluation of maltodextrin capped cadmium sulfide nanoparticles in human cell lines and chicken embryos

Our aim was to synthesize, characterize and evaluate maltodextrin coated cadmium sulfide (CdS‐MD) semiconductor nanoparticles in human cell lines and chicken embryos. The particle size and morphology of the CdS‐MD nanoparticles were characterized using TEM. Toxicological evaluation was carried out by using human tumor cell lines. Fertile chicken eggs were used for stereoscopic evaluation of growth defects in CdS‐MD nanoparticles exposed embryos. CdS‐MD nanoparticles at 5ìg/mL induced cell death by apoptosis and necrosis in MDA‐MD‐231 cells in a dose response manner. The exposure of these cells to 7–9ìg/mL of CdS‐MD nanoparticles induced ROS production. CdS‐MD nanoparticles‐treated MDA‐MB‐231 cells at >;3ìg/mL increased cell proliferation in a dose response manner at 7 days. Exposures of chicken embryos to 6ìg/mL CdS‐MD nanoparticles resulted in a dose‐dependent increase in anomalies centered on the heart, central nervous system, placodes, neural tube and somites. Our results indicate that CdS‐MD nanoparticles induce cell death and alter cell proliferation in human cell lines at concentrations higher than 3ìg/mL, and they are embryotoxic at doses of 6ìg/mL and higher.

Synthesis, characterization and toxicological evaluation of maltodextrin capped cadmium sulfide nanoparticles in human cell lines and chicken embryos

BackgroundSemiconductor Quantum dots (QDs) have become quite popular thanks to their properties and wide use in biological and biomedical studies. However, these same properties entail new challenges in understanding, predicting, and managing potential adverse health effects following exposure. Cadmium and selenium, which are the major components of the majority of quantum dots, are known to be acutely and chronically toxic to cells and organisms. Protecting the core of nanoparticles can, to some degree, control the toxicity related to cadmium and selenium leakage.ResultsThis study successfully synthesized and characterized maltodextrin coated cadmium sulfide semiconductor nanoparticles. The results show that CdS-MD nanoparticles are cytotoxic and embryotoxic. CdS-MD nanoparticles in low concentrations (4.92 and 6.56 nM) lightly increased the number of HepG2 cell. A reduction in MDA-MB-231 cells was observed with concentrations higher than 4.92 nM in a dose response manner, while Caco-2 cells showed an important increase starting at 1.64 nM. CdS-MD nanoparticles induced cell death by apoptosis and necrosis in MDA-MD-231 cells starting at 8.20 nM concentrations in a dose response manner. The exposure of these cells to 11.48-14.76 nM of CdS-MD nanoparticles induced ROS production. The analysis of cell proliferation in MDA-MB-231 showed different effects. Low concentrations (1.64 nM) increased cell proliferation (6%) at 7 days (p < 0.05). However, higher concentrations (>4.92 nM) increased cell proliferation in a dose response manner (15-30%) at 7 days. Exposures of chicken embryos to CdS-MD nanoparticles resulted in a dose-dependent increase in anomalies that, starting at 9.84 nM, centered on the heart, central nervous system, placodes, neural tube and somites. No toxic alterations were observed with concentrations of < 3.28 nM, neither in cells nor chicken embryos.ConclusionsOur results indicate that CdS-MD nanoparticles induce cell death and alter cell proliferation in human cell lines at concentrations higher than 4.92 nM. We also demonstrated that they are embryotoxic. However, no toxic effects were observed with doses lower than 3.28 nM in neither cells nor chicken embryos. The CdS-MD nanoparticles used in this study can be potentially used in bio-imaging applications. However, further studies using mammalian species are required in order to discard more toxic effects.

Efficacy and security of 3 analogues of L‐carnitine in vitro and in vivo models

The metabolic syndrome (MS) is the common denominator of the relationship between Diabetes Mellitus and cardiovascular risk. The L‐carnitine has proven to be effective in treating a variety of diseases, among them Diabetes Mellitus and hyperlipidemia. The aim was to evaluate the efficacy and security of 3 analogues of L‐carnitine. In vitro model we developed a tolerance to glucose (g) in liver cells, the cells were treated with L‐carnitine and 3 analogues, it was determinate the intracellular values of glucose, cholesterol (c) triglycerides (t) and glycogen (gy). In vivo model we developed a MS model in rats, were treated only with the analogue 3 and evaluated the serum levels of g, c and t, as well as the gy levels in liver and muscle. In vitro and in vivo assay demonstrated that analogue 3 was safe in cells and rats. The results showed a change in the intracellular levels of g, c and t in the liver cells treated with the analogue 2 and 3 and decrease in the serum levels of g and t in rats. The security evaluation did not show evidences of alterations on cell viability and proliferation in human cell lines treated with the analogues and only was observed an increase in the lymphocyte levels in rats. In conclusion, the analog 3 was effective and safe in this study. Research support CONACYT 131321.

Identification and characterization of novel potentially oncogenic mutations in the human BAF57 gene in a breast cancer patient

BAF57 is a core subunit present in all mammalian SWI/SNF ATP-dependent chromatin remodeling complexes, which regulates important biological processes including gene transcription, DNA recombination, DNA repair, and DNA replication. Among other functions, BAF57 mediates the recruitment of SWI/SNF to sequence-specific transcription factors. Thus, BAF57 plays a crucial role in regulating estrogen-dependent gene expression and proliferation in human cell lines derived from breast tumors. Increasing genetic and biochemical evidences suggest that mutations in BAF57 or alterations in its expression could play an oncogenic role in the mammary gland. Here, we describe two novel mutations in the BAF57 gene found in a breast cancer patient. Both mutations originate premature stop codons, leading to truncated proteins, structurally similar to another BAF57 mutant previously found in a human cell line derived from a breast tumor (BT-549). The expression of these novel BAF57 mutants has abnormally high estrogen receptor alpha (ERα) coactivating potential, suggesting that they might be involved in the aberrant estrogen-dependent proliferation that occur in the majority of breast tumors that retain ERα expression. In addition, the mutations in BAF57 affect its functional interaction with the androgen receptor and ETS2, two transcription factors that play an important role in breast cell biology. Therefore, mutations in BAF57 could impinge on several oncogenic signaling pathways contributing to the origin and/or development of breast cancer.

182. Regulation of PDX-1 Stability via Ubiquitination is Dependent Upon the Single Nucleotide Polymorphism (SNP) Status of Human Somatostatin Receptor Subtype 5 (SSTR5)

We have previously shown that SSTR5 is an important regulator of insulin expression/secretion and islet proliferation via pancreatic-duodenal homeobox 1(PDX-1) in mice. We have also identified a germline, non-synonymous single nucleotide polymorphism (SNP) of SSTR5 in human blood and pancreatic cancer specimens, which results in mutation of intracellular C-terminal proline 335 to leucine (SSTR5 P335L). The purpose of this study was to determine whether SSTR5 P335L SNP has any functional significance on PDX-1 expression and proliferation in human cell lines.

Cytotoxic and Apoptogenic Activity of a Methanolic Extract from the Marine Invertebrate Ciona intestinalis on Malignant Cell Lines

Marine invertebrates provide a series of natural products with different biological activities. Several of these compounds and their derivatives showed a potent anticancer effect. Tunicates represent an important source of bioactive agents, leading to the isolation of ecteinascidin-743 (ET-743), a compound isolated from the Caribbean sea squirt Ecteinascidia turbinata with a potent cytotoxic activity against a variety of tumours in vitro and in vivo. Current phase II clinical trials against soft tissue sarcomas in Europe and the United States indicate that ET-743 represents a highly promising anticancer agent. Another example is aplidine from the Mediterranean tunicate Aplidium albicans, with a broad spectrum activity against various types of cancers, such as colorectal, lymphoma, thyroid and renal cancers. In the present work, we reported, for the first time, that a partially purified methanolic extract prepared from the ascidian Ciona intestinalis inhibited cell proliferation in human cell lines of different origin, including Caco2, HPB-ALL, U-937 and HL-60 and induced early apoptotic events, such as caspase-3 activation and internucleosomal DNA degradation. We suggest the presence in the Ciona intestinalis extract of bioactive compounds possessing anticancer activity.

Pentoxifylline promotes development of murine colon adenocarcinoma-derived metastatic tumors in liver

Pentoxifylline is commonly used in the treatment of peripheral vascular diseases. It improves microcirculatory flow and tissue perfusion. Moreover, pentoxifylline displays some immunomodulatory properties that presumably might affect the anticancer response. Therefore, the aim of the present study was to evaluate the influence of pentoxifylline on tumor development. Balb/c mice were injected with murine colon adenocarcinoma C-26 cells intravenously, into the vena portae, and divided into two groups. Mice from the experimental groups received daily intraperitoneal injections of pentoxifylline (30 mg/kg) while the controls were injected with 0.9% NaCl. Two weeks after C-26 cell inoculation mice were sacrificed and autopsy was performed. It was found that the livers of control animals revealed only several small tumor foci, whereas the livers of pentoxifylline-treated mice displayed numerous cancerous outgrowths. The mean liver weight in pentoxifylline group was 2.21+/-0.62 g as compared to 1.36+/-0.15 g in controls (P=0.004). Moreover, the influence of pentoxifylline on murine and human cell line proliferation in vitro was evaluated. It has been observed that pentoxifylline, at pharmacologically achievable concentrations, stimulated the proliferation of murine (C-26) and human (CaSki, U-937) cell lines. However, it did not stimulate human melanoma (WM-35) cell proliferation. Since there has been no evidence so far that pentoxifylline may promote tumor progression, it is still considered to be a safe drug. Moreover, some beneficial properties of pentoxifylline, which could be useful in cancer treatment, have been reported and a few clinical trials with oncological patients have been performed. Surprisingly, our study revealed that pentoxifylline significantly promoted C-26-derived metastatic tumor growth in liver. Although this model might be unique in its sensitivity to tumor-promoting effects of pentoxifylline, it cannot be excluded that similar effects might occur in some cases of tumors developing in humans. Such effects could be relevant, since the stimulatory influence of pentoxifylline on proliferation does not appear to be species- or tissue-specific.

The influence of atracurium, cisatracurium, and mivacurium on the proliferation of two human cell lines in vitro.

We tested the influence of atracurium and cisatracurium (final concentrations: 0, 0.96, 3.2, 9.6, 32, and 96 microM) on proliferation of human cells (hepatoma HepG2 cells and human umbilical vein endothelial cells) in vitro. In additional experiments, glutathione, N-acetylcysteine, or carboxyl esterase was added before the addition of either relaxant. The number of cells counted after 72 h of incubation was expressed as a percentage of the mean cell number in wells incubated without additives. Atracurium and cisatracurium progressively decreased cell proliferation in a concentration-dependent pattern. With human umbilical vein endothelial cells, atracurium or cisatracurium (3.2 microM) decreased the cell count to 67.7 % (SD, 14.8%) and 50% (SD, 8.6%), respectively. Cell proliferation was not inhibited by mivacurium. The results were similar to those with HepG2 cells. Glutathione, N-acetylcysteine, and carboxyl esterase partially reversed the effects of atracurium and cisatracurium. When incubated in a buffer with glutathione, atracurium decreased the number of glutathione-sulfhydryl groups. The findings that atracurium and cisatracurium inhibit proliferation of human cell lines in vitro, but that mivacurium does not, and that this effect is alleviated by glutathione and N-acetylcysteine, as well as by the carboxyl esterase, indicate that the inhibition may be caused by the reactive acrylate metabolites.

Human ARF binds E2F1 and inhibits its transcriptional activity.

The INK4a/ARF locus which is frequently inactivated in human tumours encodes two different tumour suppressive proteins, p16(INK4a) and ARF. p16(INK4a) is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a final effector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identified. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance.

Cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro.

Acute measles is associated with pronounced immunosuppression characterized both by leukopenia and impaired lymphocyte functions. In an earlier study, we found that mitogen-dependent proliferation of uninfected human peripheral blood lymphocytes (PBLs) and spontaneous proliferation of human cell lines of lymphocytic or monocytic origin was impaired after contact with UV-inactivated, measles virus (MV)-infected cells, UV-inactivated MV or with cells transfected with MV glycoproteins (gp) F and H. We now show that mitogen-stimulated PBLs and Jurkat cell clones either highly sensitive or resistant to CD95-induced apoptosis have a similar sensitivity to MV-induced inhibition and do not undergo apoptosis. Moreover, unimpaired mitogen-dependent upregulation of important activation markers, including IL-2R, was observed in PBL cultures after contact with MV-infected, UV-irradiated presenter cells. This indicates that the cells were indeed viable and acquire a state of activation. Less IL-2 was released from PBLs after contact with MV-infected presenter cells when compared with that released after contact with uninfected cells. However, mitogen-induced proliferation of PBLs was not restored by addition of IL-2 under these conditions. It appeared that a higher fraction of mitogen-stimulated PBLs accumulated in the G0/G1 phase of the cell cycle after contact with MV-infected cells. Thus, the mitogen-unresponsiveness of PBLs seen after contact with MV-infected cells is due to cell cycle arrest rather than apoptosis.

HIV-1 gp41 selectively inhibits spontaneous cell proliferation of human cell lines and mitogen- and recall antigen-induced lymphocyte proliferation

Human immunodeficiency virus type 1 (HIV-1) transmembrane glycoprotein 41 (gp41) contains an immunosuppressive domain (Env amino acids 583–599). Previous studies by us and others using recombinant soluble gp41 (rsgp41; amino acids 539–684) and immunosuppressive peptide (1SP; a gp41 peptide, amino acids 583–599) have shown that HIV-1 gp41 by the immunosuppressive domain could bind to several proteins on human T, B and monocyte cell lines, and also to normal human peripheral blood mononuclear cells. In this study we demonstrated that HIV-1 rsgp41 could inhibit spontaneous cell proliferation of human T cell lines H9 and Jurkat, B cell lines Raji and Daudi, monocyte cell line U937, but could not inhibit cell proliferation of human fibroblast cell line HEF and green monkey kidney cell line Cos-1. HIV-1 rsgp41 could inhibit also concanavalin A (Con A)-, phytohaemagglutinin (PHA)- and tetanus toxoid (TT)-induced cell proliferation of normal human peripheral blood lymphocytes, with 50% inhibition at a concentration of 8 μM, but could not inhibit pokeweed mitogen (PWM)-induced lymphocyte proliferation. Furthermore, recombinant soluble gp36 of HIV-2 like HIV-1 rsgp41 could inhibit Con A-, but not PWM-induced lymphocyte proliferation. These results indicate that HIV-1 gp41-induced inhibition of proliferation is selective in so far as the effect of PWM is not altered while the effects of several other stimuli are.

Downregulation of c-mycExpression by Tumor Necrosis Factor-α in Combination with Transforming Growth Factor-β or Interferon-γ with Concomitant Inhibition of Proliferation in Human Cell Lines

The modulation of cell growth by tumor necrosis factor-alpha (TNF-alpha), or TNF-alpha in combination with transforming growth factor-beta (TGF-beta) or interferon-gamma (IFN-gamma) was investigated. TNF-alpha inhibited the proliferation of U937 cells, a monocytic leukemic cell line, and of NA cells that were established from oral squamous cell carcinoma. TNF-alpha showed a cytolytic effect on NA cells in the presence of actinomycin D. TNF-alpha in combination with TGF-beta and TNF-alpha combined with INF-gamma synergistically inhibited the cell proliferation of U937 and NA cells, respectively. TNF-alpha dose-dependently reduced c-myc mRNA expression of U937 and NA cells within 1 h. The combination of TNF-alpha and TGF-beta in U937 cells and that of TNF-alpha and IFN-gamma in NA cells cooperatively reduced the expression of c-myc mRNA. TNF-alpha had little or no effect on the half-life of c-myc mRNA, indicating that c-myc mRNA expression was reduced at transcriptional level. Cycloheximide did not mediate the inhibition of c-myc gene expression, suggesting that the TNF-alpha action was independent of de novo protein synthesis. These data suggest that the reduction of c-myc gene at transcriptional level by TNF-alpha or TNF-alpha in combination with TGF-beta or IFN-gamma plays a primary role in the inhibition of cell growth at an early stage.