Significant Inhibition Of DNA Synthesis Research Articles

CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 Causes Developmental Deficits in Human Cortical Neurons Derived from Induced Pluripotent Stem Cells.

The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.

Abstract 3069: The S-phase checkpoint function of MGMT provides an unexpected rational route to profoundly increase the efficacy of anticancer antimetabolites in cell culture and xenograft models

Abstract MGMT is an anti-mutagenic DNA repair protein highly expressed not only in brain tumors, but also breast cancers, melanoma and colon cancers, and confers drug resistance. Recently, we showed that human MGMT has non-repair functions with vital roles in cell cycle; MGMT underwent PCNA-dependent degradation in the S-phase along with p21cip1 and CDT1 proteins. MGMT inactivation by O6-benzylguanine (BG) or a pool of shRNAs in asynchronous or G1/S-phase synchronized cells led to the activation of a checkpoint resulting in a significant inhibition of DNA synthesis and greatly diminished progression of cells into S-phase. We hypothesized that the replication stress caused by MGMT inactivation will provide a means to combine the S-phase specific drugs for synergistic cytotoxicity. We screened the sensitivity of a panel of colon cancer and glioblastoma cell lines with this combination using MTT and clonogenic growth assays. Annexin V/PI and acridine orange/ EtBr cell stainings were used to quantify apoptosis. MGMT inhibition/depletion significantly reduced the IC50 (5 to 10-fold) for different antimetabolites (5-FU, gemcitabine, Ara-C) and enhanced the overall apoptotic cell death (>5-fold). Nevertheless, the synergistic effects strongly depended on time and administration schedule of these two agents. Thus, a pretreatment of BG or MGMT shRNA followed by 5-FU for or a simultaneous treatment of the two were far less cytotoxic than the antimetabolite first followed by BG or shRNA as in the post-treatment mode. While the pre or simultaneous MGMT inhibition significantly prolonged the antimetabolite-induced S-phase arrest, the BG-post-treatments resulted in the abrogation of antimetabolite-induced cell cycle block and rapid progression into premature mitosis and apoptosis. We observed an increased activation of checkpoint kinases (pChk1 or pChk2) during pre or simultaneous treatments. In contrast, the post-MGMT inhibition interfered with S-phase checkpoint activity and had an abnormal CDK2 activation. Finally, we verified the therapeutic potentiation of antimetabolites by BG using the HT29-luc2 subcutaneous xenografts developed in nude mice. BG (60mg/Kg), 5-FU (30 mg/Kg), gemcitabine (100 mg/Kg) and Ara-C (40 mg/Kg/dose) were administered through i.p. route once a week for 7 weeks. Tumor growth revealed a 50 to 70% more reduction in combination groups (antimetabolites+BG) compared with drugs alone. No significant changes in mouse body weight, liver functions, and whole blood counts were noted. A marked downregulation of ki-67 and increased levels of apoptotic markers (cleaved caspase-3, cleaved PARP) in was evident in tumor tissues. Collectively, our data suggest that MGMT inhibition is a rational strategy not only for alkylating agents but also for antimetabolites as well (supported by CPRIT RP130266 & RP170207 grants to KSS). Citation Format: AGM Mostofa, Debasish Basak, Hanumantha Madala, Surendra R. Punganuru, Kalkunte S. Srivenugopal. The S-phase checkpoint function of MGMT provides an unexpected rational route to profoundly increase the efficacy of anticancer antimetabolites in cell culture and xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2017-3069

C/EBPb Is a Critical Mediator of Resistance to IMiD® Immunomodulatory Compounds and Affected by IMiD Compounds Via Control of Protein Translation

AbstractAbstract 135 Background:Lenalidomide and pomalidomide are IMiD® immunomodulatory compounds that have been shown to be highly active in the treatment of multiple myeloma (MM). IMiD compounds exert their anti-tumor effects via acting on costimulatory proteins of T cells and NK cells, augmenting both the adaptive and innate immune system. But the mechanisms by which IMiD compounds directly inhibit MM cell proliferation are still unclear. Here we focused on the direct effects of IMiD compounds alone on MM cells. Results and Methods:We found that IMiDs, at concentrations as low as 0.01 μ M, induce significant inhibition of DNA synthesis in MM cells as shown by thymidine uptake. Since our previous work demonstrated that C/EBPβ is an important transcription factor which controls the growth and proliferation of myeloma cells, we analyzed the effects of IMiD compounds on C/EBPβ. We found that both pomalidomide and lenalidomide significantly decreased the protein level of C/EBPβ LAP-isoform in MM cell lines and primary MM cells. IMiD compound-induced suppression of C/EBPβ protein expression led to impaired transcription of the downstream IRF4, and subsequently to downregulation of BLIMP1 and XBP1, which are all critical for MM survival. To confirm our findings in vivo, we analyzed IRF4 expression by double labeling (IRF4+/CD138+) immunohistochemical staining of bone marrow biopsy samples of 23 myeloma patients prior to therapy and during therapy with lenalidomide. During lenalidomide therapy, the bone marrow MM cells showed a significantly weaker staining intensity for IRF4 in comparison to prior therapy. This was quantified by a significant (p<0.001) decrease of the staining score from 176 to 152, respectively. To confirm the critical role of C/EBPβ in MM we stably overexpressed C/EBPβ in MM cells. Overexpression of C/EBPβ prevented IMiD compound-induced inhibition of MM cell proliferation, indicating that C/EBPβ is critical in mediating resistance to IMiD compounds. This was supported by the fact that C/EBPβ was not down regulated in IMiD-resistant cell lines by IMiD treatment. Dissection of the C/EBPβ protein regulation revealed that IMiD compounds shut down C/EBPβ protein translation by decreasing eIF-4E. Knockdown experiments of eIF-4e resulted in downregulation of C/EBPβ, suggesting that C/EBPβ is under translational control in MM. Conclusions:Our studies, for the first time, provide evidence that IMiD compounds inhibit MM cell proliferation and survival by affecting the translation of C/EBPβ and subsequently multiple downstream transcription factors including IRF4, BLIMP1 and XBP1. Due to the critical role of C/EBPβ in mediating effects of IMiD compounds in MM, it might be a target to overcome drug resistance to IMiD compounds. The fact that pomalidomide can overcome resistance to lenalidomide in MM requires still further evaluation. Disclosures:Schafer:Celgene Corporation: Employment, Equity Ownership. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.

Bisindenoisoquinoline Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}propane bis(trifluoroacetate) (NSC 727357), a DNA Intercalator and Topoisomerase Inhibitor with Antitumor Activity

Indenoisoquinolines are topoisomerase (Top) I inhibitors developed to overcome some of the limitations of camptothecins and expand their anticancer spectrum. Bis-1,3-{(5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline)-6-propylamino}-propane bis(trifluoroacetate) (NSC 727357) is a novel dimeric indenoisoquinoline derivative with potent antiproliferative activity in the NCI-60 cell line panel, promising hollow fiber activity (score of 32) and activity against xenografts. Submicromolar concentrations of the bisindenoisoquinoline NSC 727357 induce Top1 cleavage complexes at specific sites in biochemical assays. At higher concentrations, inhibition of Top1 catalytic activity and DNA intercalation is observed. NSC 727357 also induces a limited number of Top2-DNA cleavage complexes. In contrast to the effect of other Top1 inhibitors, cells treated with the bisindenoisoquinoline NSC 727357 show an arrest of cell cycle progression in G(1) with no significant inhibition of DNA synthesis after a short exposure to the drug. Moreover, unlike camptothecin and the indenoisoquinoline MJ-III-65 (NSC 706744, 6-[3-(2-hydroxyethyl)aminopropyl]-5,6-dihydro-5,11-diketo-2,3-dimethoxy-(methylenedioxy)-11H-indeno[1,2-c]isoquinoline hydrochloride), the cytotoxicity of bisindenoisoquinoline NSC 727357 is only partially dependent on Top1 and p53, indicating that this drug has additional targets besides Top1 and Top2.

DNA topoisomerase II is required for the G0-to-S phase transition in Drosophila Schneider cells, but not in yeast.

We previously reported that DNA topoisomerase II (topo II) is required for the G(0)-to-S phase transition in mammalian cells [Hossain et al. (2002) ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells. Genes Cells 7, 285-294]. In this study, we examined whether the requirement for topo II is evolutionarily conserved in Drosophila and yeast. ICRF-193, a catalytic inhibitor of topo II, inhibited DNA synthesis in Drosophila Schneider cells released from the G(0) (stationary) phase, whereas the drug did not inhibit DNA synthesis in Schneider cells released from the M phase. Depletion of topo II mRNA by RNA-interference (RNAi) in G(0)-phase Schneider cells resulted in significant inhibition of DNA synthesis after release from G(0)-arrest. In the yeast topo II temperature-sensitive (ts) mutant, the initial cycle of DNA synthesis occurred at a restrictive temperature after release from starvation-induced G(0) phase and doubling of the DNA content in the cells was confirmed by both flow cytometry and fluorescence spectrophotometry. DNA synthesis in yeast cells after release from the G(0) phase was also observed in the presence of ICRF-193. Doubling of the DNA content was observed during spore germination of topo II ts mutant yeast at a restrictive temperature as determined by fluorescence spectrophotometry. These results indicate that topo II is required for the G(0)-to-S phase transition in Drosophila Schneider cells, but not in yeast.

Expression of somatostatin receptor mRNA in human meningiomas and their implication in in vitro antiproliferative activity.

Somatostatin receptors (SSTRs) have been detected in many normal and malignant tissues. This wide expression has been used for diagnostic, prognostic and therapeutic purposes. Five SSTR subtypes (SSTR 1-5) have been identified whose activation is responsible for the signal transduction through many different intracellular pathways. In the present study the expression of SSTR mRNA was determined by reverse-transcriptase (RT)-PCR in 42 meningiomas. About 88% of the tumors analyzed (37/42) were positive for at least one of the five SSTR subtypes displaying a variable pattern of expression of the different SSTR subtypes. SSTRI and SSTR2 were the most frequently mRNA detected (69% and 79% of the sample analyzed, respectively). The other subtypes were found in the 43%, 33% and 33% of cases for SSTR3, SSTR4 and SSTR5, respectively. In 22, out of 42 patients (52%) three or more SSTRs were detected. The expression of the different SSTR subtypes did not correlate with the expression of bcl-2 (apoptosis-associated protein) and MIB-1 (a proliferation marker), assessed by immunohistochemistry in a series of 34 tumor samples, while a correlation between the expression of SSTR3 and p53 was observed (p = 0.08). To evaluate a possible role of SSTR in the control of human meningioma cell proliferation, seven primary cell cultures obtained from fresh meningioma surgical tissues, were analyzed for their proliferative behavior by MTT assay and for their response to SST by [3H]-thymidine incorporation. In four out of six tumors (in one case no SSTR were detected) the treatment with SST caused a significant inhibition of DNA synthesis induced by the tumor-promoter phorbol myristate acetate. The evidence of the expression of SSTRs, mainly of SSTR2, in this series of specimens we analyzed altogether with in vitro antiproliferative effects of SST may open interesting perspectives for the diagnosis and the therapy of meningiomas.

Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

In vitro lead-induced cell toxicity and cytoprotective activity of fetal calf serum in human fibroblasts.

The underlying mechanisms by which lead ions produce their deleterious effects prior to the onset of clinical symptoms are incompletely understood. This study aimed to assess lead-induced cell toxicity mechanisms by focusing on the effects of the metal on cell growth, DNA synthesis, cellular ATP, intracellular hexosaminidase activity and lysosomal function, and examine the possible cytoprotective role of fetal calf serum (FCS). Several human dermal cultured fibroblast lines were exposed to Pb (400 microM) for 1-6 days with 2, 5, and 10% FCS. The earliest toxic effect of Pb was significant inhibition of DNA synthesis after 24 h direct exposure; this harmful effect was not progressive during the first 3 days, but worsened clearly on the 4th day regardless of the FCS concentration. Atime-dependent depletion of intracellularATP content was also caused by ionic lead, thereby compromising the cell energy charge which precedes cell death. Fibroblast growth was progressively and significantly inhibited from day 2 onwards; the greatest noxious effect was observed in the presence of 2% FCS: 49% reduction in cell proliferation after 5 days. Lead salts produced loss of cell adhesion to the culture dish which worsened from the 2nd day and was more pronounced when FCS in growth medium was decreased. Toxic actions on lysosomal membrane integrity provoked a decrease in neutral red uptake (NRU) which was exposure time-dependent and more marked with 2% FCS. In contrast, increased relative NRU (to 20% at 4 days), suggestive of endocytosis-induced lysosome enlargement, was observed in Pb-exposed cells. Intracellular hexosaminidase activity was not negatively affected until 5 days after exposure to Pb salts. FCS had a significant cytoprotective effect on Pb-induced toxicity.

Developmental Neurotoxicity of Chlorpyrifos Modeled in Vitro: Comparative Effects of Metabolites and Other Cholinesterase Inhibitors on DNA Synthesis in PC12 and C6 Cells

The widely used organophosphate pesticide chlorpyrifos is a suspected neuroteratogen. In the current study, we compared the effects of chlorpyrifos and its major metabolites in two in vitro models, neuronotypic PC12 cells and gliotypic C6 cells. Chlorpyrifos inhibited DNA synthesis in both cell lines but had a greater effect on gliotypic cells. Chlorpyrifos oxon, the active metabolite that inhibits cholinesterase, also decreased DNA synthesis in PC12 and C6 cells with a preferential effect on the latter. Trichloropyridinol, the major catabolic product of chlorpyrifos, had a much smaller, but nevertheless statistically significant, effect that was equivalent in both cell lines. Diazinon, another organophosphate pesticide, also inhibited DNA synthesis with preference toward C6 cells, but was less effective than was chlorpyrifos. Physostigmine, a non-organophosphate cholinesterase inhibitor, was less effective than either chlorpyrifos or diazinon, but still caused significant inhibition of DNA synthesis in C6 cells. We also found that the addition of sera protected the cells from the adverse effects of chlorpyrifos and that the effect could be reproduced by addition of albumin. These results indicate that chlorpyrifos and other organophosphates such as diazinon have immediate, direct effects on neural cell replication, preferentially for gliotypic cells. In light of the protective effect of serum proteins, the fact that the fetus and newborn possess lower concentrations of these proteins suggests that greater neurotoxic effects may occur at blood levels of chlorpyrifos that are nontoxic to adults.

Abstracts of literature

Abstracts of literature

Folic acid inhibits homocysteine-induced proliferation of human arterial smooth muscle cells

Purpose: An elevated plasma homocysteine level has been identified as an independent risk factor for atherosclerosis. Whether this represents a marker for vascular disease or a direct effect on the vasculature remains unclear. Because vascular smooth muscle cells (VSMCs) play an integral role in the atherosclerotic process, we studied the effect of homocysteine on human infragenicular VSMC proliferation and the role of folic acid in reversing the homocysteine effect. Methods: Human infragenicular VSMCs harvested from amputation specimens were studied. Various cell groups were exposed to physiologic (6.25 μmol/L and 12.5 μmol/L) and pathologic (25 μmol/L to 500 μmol/L) concentrations of homocysteine. Similar groups were simultaneously exposed to 20 nmol/L of folic acid. Cell counts and DNA synthesis, as reflected by [methyl- 3H]-thymidine incorporation, were performed at 6 days and 24 hours, respectively. Additional groups were exposed to various combinations of folic acid (20 nmol/L), vitamin B 6 (145 nmol/L), and vitamin B 12 (0.45 nmol/L) in the presence of homocysteine (25, 50, and 250 μmol/L). Results: Homocysteine resulted in a dose-dependent increase in DNA synthesis and cell proliferation. Cell counts increased significantly at homocysteine concentrations ranging from 25 μmol/L to 500 μmol/L ( P < .05), with a maximal increase of 98% at 500 μmol/L of homocysteine. The addition of 20 nmol/L folic acid resulted in significant inhibition of cell proliferation at all homocysteine concentrations studied ( P < .001). Maximal inhibition of 70% occurred in the cells exposed to 50 μmol/L of homocysteine. The increases in [methyl- 3H]-thymidine incorporation ranged from 36% at 6 μmol/L homocysteine to a maximum of 247% at 500 μmol/L homocysteine. All increases were statistically significant ( P < .05). The addition of 20 nmol/L folic acid resulted in significant inhibition of DNA synthesis ( P < .002). Vitamins B 6 and B 12 did not demonstrate significant antiproliferative properties. Conclusion: A possible role of homocysteine in the formation of atherosclerotic lesions is through a direct proliferative effect on VSMCs in a dose-dependent fashion. Folic acid intake at levels available in dietary supplements may prove protective in hyperhomocysteinemia-induced atherosclerosis. Vitamins B 6 and B 12 alone do not appear to exhibit a substantial inhibitory effect in the setting of elevated homocysteine levels. (J Vasc Surg 1999;30:1121-8.)

Transforming Growth Factor-α and Insulin-like Growth Factor-I, but Not Epidermal Growth Factor, Elicit Autocrine Stimulation of Mitogenesis in Endometrial Cancer Cell Lines

Objectives.Endometrial carcinoma cell lines were evaluated for epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and insulin-like growth factor I (IGF-1) production and for autocrine stimulation.Methods.Conditioned, serum-free media (CM) from cell lines RL95-2, KLE, HEC, and Ishikawa (ISH) were concentrated radioimmunoassayed (RIA). Samples for the IGF-1 assay were extracted with acid–ethanol to remove IGF-1 binding protein. Polymerase chain reaction (PCR) was used to validate the presence of mRNA for growth factors and receptors. Cells were incubated with Ab528, an antibody blocking EGF receptors, and αIR3, an antibody blocking IGF-1 receptors. Proliferation was quantified using [3H]thymidine incorporation.Results.TGF-α was detected in CM: RL95-2 (0.4 ± 0.001 ng/ml), KLE (0.7 ± 0.003 ng/ml), HEC (0.8 ± 0.01 ng/ml), ISH (1.2 ± 0.05 ng/ml). No EGF was detected in CM. In extracted samples, IGF-1 was detected in CM: RL95-2 (0.8 ± 0.03 ng/ml), KLE (1.25 ± 0.02 ng/ml), HEC (1.6 ± 0.01 ng/ml), ISH (1.6 ± 0.08 ng/ml). Unconditioned media served as the control. EGF, TGF-α, and IGF-1 mRNA was identified in all cell lines, as was the mRNA for EGF and IGF-1 receptors. Incubation with Ab528 or αIR3 resulted in significant inhibition of DNA synthesis in HEC 1A, KLE, and ISH. No inhibition was detected in the RL95-2 cell line. A control antibody did not inhibit the cell lines.Conclusion.Autocrine production and stimulation of endometrial carcinoma cell lines by TGF-α and IGF-1 are demonstrated in three of four endometrial cancer cell lines. No measurable EGF was produced by any of the cell lines.

Pyran derivatives XX. 2-Aminochromone benzo-fused derivatives with antiproliferative properties

The N-substituted 2-aminochromones 1 and their benzo-fused derivatives 2–4 described herein were mostly prepared by treating the corresponding (methylthio) derivatives 10–13 with an excess of the proper amines. Only the morpholino derivatives 3d and 4c were obtained from the reaction of the ethyl 3-morphohno-3-oxopropanoate/POCl3 reagent with 1-naphthol or 1-methyl-2-naphthol, respectively. The amino derivatives 1–4, as well as their methylthio analogues 10–13, were tested in vitro for their inhibitory activity on the infectivity of T2 bacteriophage, on the macromolecular synthesis in Ehrlich cells and on the clonal growth capacity of HeLa cells. Several of the angular or linear aminonaphthopyranones 2 and 3 or 4, respectively, and the (methylthio) derivatives 10, 11 and 13 induced a significant inhibition of DNA synthesis, but usually a clearly lower inhibition of clonal growth. Only the linear 2-amino- 10-methyl-4H-naphtho[2,3-b]pyran-4-ones 4a and 4b significantly inhibited the clonal growth in HeLa cells and T2 bacteriophage infectivity, respectively.

Skin Cancer Chemopreventive Effects of a Flavonoid Antioxidant Silymarin Are Mediated via Impairment of Receptor Tyrosine Kinase Signaling and Perturbation in Cell Cycle Progression

Enhanced tyrosine kinase activity due to aberrant or overexpression of receptor and/or non-receptor tyrosine kinases has been implicated in a variety of human malignancies including cutaneous neoplasms. Epidermal growth factor receptor (EGFR)-mediated tyrosine phosphorylation may be a primary indicator of signal transduction regulating cell growth and proliferation. Recent studies have shown that skin tumor promoters such as phorbol ester and ultraviolet B radiation activate EGFR in mouse skin as well as in cell culture. Similarly, oxidative stress, which is implicated in skin tumor promotion, also activates EGFR-mediated cell signaling. Since this signaling pathway has been suggested to be involved in skin tumor promotion, its impairment by antioxidants may lead to an efficient way for skin cancer prevention and therapy. Recently, we showed that silymarin, a flavonoid antioxidant, affords exceptionally high to complete protection against several skin tumor promoters caused tumor promotion in mouse skin. Employing human epidermoid carcinoma cells A431 that contain overexpressed EGFR, in this study, we assessed whether the anti-skin tumor promoting effects of silymarin are due to its inhibitory effect on EGFR activation and down stream signaling pathway leading to perturbations in cell cycle progression. Treatment of cells with silymarin resulted in a significant inhibition of ligand-induced activation of EGFR with no change in its protein levels. Silymarin treatment also resulted in a significant decrease in tyrosine phosphorylation of Shc, an immediate downstream target of EGFR, but no change in the protein levels of Shc. The inhibition of EGFR activation by silymarin was associated with a highly significant to complete inhibition of EGFR intrinsic kinase activity. Cells treated with silymarin also showed a significant G2-M arrest in cell cycle progression, and a highly significant inhibition of DNA synthesis and cell growth in a dose-dependent manner. Taken together, these results suggest that skin cancer chemopreventive effects of silymarin are mediated via impairment of EGFR signaling which ultimately leads to perturbation in cell cycle progression resulting in the inhibition of proliferation and induction of growth arrest.

Inhibition of the ABL Kinase Activity Blocks the Proliferation of BCR/ABL+Leukemic Cells and Induces Apoptosis

ABSTRACT: The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL bothin vitroandin vivoand to inhibit the growth of v-abl and bcr/abl transfectants, as well as thein vitroformation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34+cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. Thein vivophosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies.All six BCR/ABL+lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations ≤3 μM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too.The activity of CGP57148B can be monitored inex vivoisolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected.These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and fibroblast-derived cells will have to be closely monitored. Thein vivomonitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.

Molecular Delivery System for Antisense Oligonucleotides: Enhanced Effectiveness of Antisense Oligonucleotides by HVJ-liposome Mediated Transfer.

BACKGROUND: The effectiveness of antisense oligodeoxynucleotides for in vitro and in vivo studies is limited by a low efficiency of cellular uptake and instability due to degradation by nucleases. To overcome some of these problems, we recently developed a transfer method that utilizes inactivated Sendai virus (hemagglutinating virus of Japan [HVJ]) complexed with liposomes to deliver antisense oligodeoxynucleotides. In this study, we compared the effectiveness of the HVJ-liposome method versus a cationic liposome method versus passive uptake to deliver antisense oligodeoxynucleotides against basic fibroblast growth factor on angiotensin (Ang) II-induced rat vascular smooth muscle cell growth. METHODS AND RESULTS: Twenty to twenty-eight hours after transfection, antisense fibroblast growth factor oligodeoxynucleotides introduced by passive uptake and HVJ-liposome method decreased basal DNA synthesis significantly as compared to the sense, control, and scrambled oligodeoxynucleotides groups; however, 60-68 hours after transfection, only antisense fibroblast growth factor oligodeoxynucleotides transduced by the HVJ-liposome method resulted in a significant inhibition of DNA synthesis under basal and Ang II-(10(-;6)M) stimulated conditions. The IC(25) of oligodeoxynucleotides assessed by the inhibition of thymidine incorporation was significantly lower using the HVJ-liposome method than those using the other transfer methods. To clarify the mechanisms of cellular uptake of oligodeoxynucleotides with the HVJ-liposome method, we studied the cellular fate of fluorescein isothiocyanate (FITC)-labeled oligodeoxynucleotides FITC-oligodeoxynucleotides was localized in nuclei at 5 minutes after transfection with the HVJ-liposome method. In contrast, FITC-oligodeoxynucleotides introduced by passive uptake was detected in nonnuclear cellular compartments, possibly endosomes, but not the nuclei. Cellular fluorescence of oligodeoxynucleotides introduced by passive uptake disappeared within 24 hours, while that introduced by the HVJ-liposome method could be observed up to 72 hours. CONCLUSION: These results demonstrate that the HVJ-liposome transfer enhanced the effectiveness of AS-fibroblast growth factor via its specific molecular mechanisms of transfer.

Inhibition of DNA synthesis by paracetamol in different tissues of the rat in vivo

DNA synthesis in the spleen, testis, thymus, stomach, small intestine and bone marrow was inhibited by 70–90% at 1 h following an oral dose of paracetamol (1 g/kg). This inhibitory effect was still apparent using a lower dose of 125 mg/kg paracetamol, but not when the dose was reduced to 60 mg/kg. In contrast, the liver was resistant to the inhibitory action of paracetamol on DNA synthesis, there being no significant inhibition of DNA synthesis at 500 mg/kg or 1 g/kg paracetamol. These doses and the associated plasma levels are in the range found in human overdose. Tissue levels of paracetamol in the liver, spleen, thymus, kidney and testis were essentially the same as the plasma level. However the apparent paracetamol tissue levels in the stomach wall and duodenum were orders of a magnitude higher than the plasma level. The tissue levels of paracetamol did not explain the differences between tissues in the degree of inhibition of DNA synthesis, in particular the high levels of paracetamol in the tissue of the stomach and duodenum did not result in higher levels of inhibition in these tissues. This study also shows that the inhibitory effect of paracetamol on DNA synthesis is transient. All the tissues, except the spleen, no longer showed inhibition of DNA synthesis by 4 h post paracetamol dosing.

Developmental Neurotoxicity of Chlorpyrifos: Cellular Mechanisms

Chlorpyrifos, one of the most widely used pesticides, exhibits greater toxicity during development than in adulthood. We administered chlorpyrifos to neonatal rats in apparently subtoxic doses that caused no mortality and little or no weight deficits and examined developing brain regions (cerebellum, forebrain, brainstem) for signs of interference with cell development. One-day-old rats given 2 mg/kg sc of chlorpyrifos showed significant inhibition of DNA synthesis in all brain regions within 4 hr of treatment; equivalent results were obtained when a small dose (0.6 μg) was introduced directly into the brain via intracisternal injection, indicating that the actions were not secondary to systemic toxicity. Inhibition of DNA synthesis was also seen at 8 days of age; however, at this point, there was regional selectivity, with sparing of the cerebellum. Between 1 and 8 days of age, brain regions develop wide disparities in cholinergic innervation; accordingly, we tested whether the effect of chlorpyrifos was mediated through cholinergic actions on nicotinic receptors known to mediate inhibition of DNA synthesis. Pretreatment with mecamylamine caused a decline in DNA synthesis by itself, but nevertheless prevented the effect of chlorpyrifos. Additionally, chlorpyrifos administration at 1 day of age caused an even larger inhibition of protein synthesis throughout the brain; the effect was distinct from that on DNA synthesis, as it diminished substantially by 8 days of age and did not develop any regional selectivity. The effects of chlorpyrifos on DNA and protein synthesis were not secondary to generalized cell damage or suppression of cell metabolism, as evidenced by maintenance of normal ornithine decarboxylase activities. These results indicate that low doses of chlorpyrifos target the developing brain during the critical period in which cell division is occurring, effects which may produce eventual cellular, synaptic, and behavioral aberrations after repeated or prolonged subtoxic exposures.

Functional role of CD26 on human B lymphocytes

CD26 is a well-known activation marker on T cells and natural killer (NK) cells [1]. It is identical with the ectopeptidase dipeptidyl peptidase IV (DP IV). The expression of CD26 on B cells has been discussed controversially [2,3]. We have studied the expression of this enzyme on B cells from the peripheral blood of healthy donors and of CVID patients, on cells of the Daudi Burkitt line and the EBV-transformed B-cell lines Jojo and Laz509. DP IV was detected by using anti-CD26 monoclonal antibodies and with help of specific enzyme substrates. Further the influence of specific synthetic DP IV inhibitors on mitogenic activation of purified B cells and DNA synthesis of cell lines was studied. We could show that in both groups 0–5% of freshly isolated CD20-positive B cells do express the CD26 antigen. After stimulation with pokeweed mitogen or St. aureus protein, the fraction of CD26-positive cells was enhanced up to 51% and 36%, respectively. Interestingly, induction of CD26 expression on B cells from CVID patients occurs in a manner similar to the B cells from healthy donors. Treatment of peripheral blood B cells and B-cell lines with highly specific competitive DP IV inhibitors leads to a significant inhibition of DNA synthesis in a dose-dependent manner. These data show that CD26 can be considered to be an activation marker not only of T- and NK cells but also of a main population of B cells, suggesting an involvement of CD26 in B-cell activation.

Protein kinase C pathway and proliferative responses of aged and young rat vascular smooth muscle cells

Alterations of vascular smooth muscle cell (VSMC) proliferation have been implicated in the age-dependent susceptibility to atherosclerosis. Although it is known that protein kinase C (PKC) is involved in the mechanism of VSMC proliferation, there are no data on the possible involvement of PKC in disregulating VSMC proliferation in aged vascular cells. We evaluated the proliferative pattern, the PKC responsiveness and the effect of phorbol ester (PMA) treatment on vascular cell growth and cell cycle distribution in VSMCs from young and aged rats. The proliferative response was significantly higher in aged than in young cells after serum stimulation (7.5 vs. 2.8 × 10 4, 18 vs. 12 x 10 4, 26 vs. 22 × 10 4 cells/well, aged vs. young at days 2, 4, 6; P < 0.005). On the contrary, aged cells showed a significant inhibition of DNA synthesis at 48 h incubation with PMA concentrations of 1, 10, 100 nM (−47%, −53%, −58%, respectively) compared with controls (fetal calf serum 0.5%) and cell count (average decrease: −38% from 48 h to 96 h) after treatment with PMA 10 nM. The opposite was observed in young cells on [ 3H]thymidine incorporation with PMA 1, 10, 100 nM (+52%, +100%, +121%, respectively and cell count (average increase +55% from 48 h to 96 h). In addition, inhibition of the cell cycle from G 1 to the S phase and reduction of PKC translocation in aged VSMC were observed. Alterations of PKC function could be involved in the disregulation of aged VSMC proliferation, which seems to characterize the increased susceptibility to atherosclerosis.