Evidence Of Cell Cycle Arrest Research Articles

Interferome perturbation of human brain organoids induces progenitor and neuronal dysfunction seen in multiple sclerosis and autism (P2-3.015)

<h3>Objective:</h3> To characterize the effect of activation of the interferome in human organoids toward understanding human cortical neuronal dysfunction. <h3>Background:</h3> The mechanisms by which maternal infection and neuroinflammation impact human brain development, function, and repair remain unclear. IFN-g is a proinflammatory cytokine found elevated in serum of pregnant mothers with children diagnosed with autism and in Th1 cells in the cerebrospinal fluid of multiple sclerosis patients. <h3>Design/Methods:</h3> Nearly three hundred human cerebral organoids derived from iPSCs were used across all analyses. Organoids were continually exposed to IFN-g and compared to controls for up to 80 days in culture. 10X single-cell transcriptomics was performed at three timepoints, along with immunolabeling and pulse EdU experiments. Bioinformatic and Markov Chain analyses were used to determine the molecular, cellular, and systems level effects of IFN-g. <h3>Results:</h3> IFN-g exposure at low concentration restricts organoid growth across timepoints, diminishes the pool of cycling radial glia progenitors, and disrupts ventricular adherens cytoarchitecture. We find no evidence of cell cycle arrest or apoptosis, but rather an induction of premature neuronal differentiation and acceleration of neuronal maturation, that stems from cell-type-specific transcriptional rewiring by IFN-g, inducing expression of inflammatory and neurogenic genes that persists in all cell lineages. Markov chain modeling demonstrates that IFN-g targets differentiation lineages in a parallel pan-neuronal fashion. Finally, we find that cell-type-specific gene dysregulation induced by IFN-g overlaps with patient derived organoids from genetically linked autism spectrum disorder, and neuronal intrinsic dysregulation observed in cortical neurons in progressive multiple sclerosis. <h3>Conclusions:</h3> Our results show the feasibility of using a human brain organoid to model the effects of neuroinflammation on human brain development and function. This approach reveals a novel inflammatory mechanistic link with autism and identifies a critical network of neuronal identity and synaptic genes perturbed in multiple sclerosis recapitulated in a preclinical model. <b>Disclosure:</b> Ethan Hollingsworth has nothing to disclose. Mr. Julian has nothing to disclose. Fumihiro Watanabe has nothing to disclose. Mr. Reutershan has nothing to disclose. Miss Julian has nothing to disclose. Ms. Martorell Serra has nothing to disclose. Sofia Lizarraga has nothing to disclose. Dr. Hester has nothing to disclose. Dr. Imitola has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis . The institution of Dr. Imitola has received research support from biogen. Dr. Imitola has a non-compensated relationship as a Board Member with National MS Society that is relevant to AAN interests or activities. Dr. Imitola has a non-compensated relationship as a Committee Member with International Society for Stem Cell Research that is relevant to AAN interests or activities.

Selective Anticancer and Antimicrobial Metallodrugs Based on Gold(III) Dithiocarbamate Complexes

New dithiocarbamate cycloaurated complexes have been synthesized and their physicochemical and in vitro antitumor properties have been evaluated. All the performed studies highlighted good transport through the blood and biodistribution, according to the balance between the properties of hydrophilicity/lipophilicity and the binding of moderate strength to the BSA protein. Furthermore, none of the complexes exhibited reduction or decomposition reactions, presenting excellent physiological stability. The in vitro cytotoxic effect was evaluated on human colon cancer cell line Caco-2/TC7, and the complexes showed great antiproliferative activity and excellent selectivity, as much less effect was detected on normal Caco-2/TC7 cells. Most of the complexes exhibit antiproliferative activity that was better than or similar to auranofin, and at least nine times better than that of cisplatin. Its action mechanism is still under discussion since no evidence of cell cycle arrest was found, but an antioxidant role was shown for some of the selective complexes. All complexes were also tested as antimicrobial drugs, exhibiting good activity towards S. aureus and E. coli. bacteria and C. albicans and C. neoformans fungi.

Liposomes Co-Encapsulating Cisplatin/Mifepristone Improve the Effect on Cervical Cancer: In Vitro and In Vivo Assessment.

Cervical cancer is usually diagnosed in the later stages despite many campaigns for early detection and continues to be a major public health problem. The standard treatment is cisplatin-based chemotherapy plus radiotherapy, but patient response is far from ideal. In the research for new drugs that enhance the activity of cisplatin, different therapeutic agents have been tested, among them the antiprogestin mifepristone. Nevertheless, the efficacy of cisplatin is limited by its low specificity for tumor tissue, which causes severe side effects. Additionally, cervical tumors often become drug resistant. These problems could possibly be addressed by the use of liposome nanoparticles to encapsulate drugs and deliver them to the target. The aim of this study was to prepare liposome nanoparticles that co-encapsulate cisplatin and mifepristone, evaluate their cytotoxicity against HeLa cells and in vivo with subcutaneous inoculations of xenografts in nu/nu mice, and examine some plausible mechanisms of action. The liposomes were elaborated by the reverse-phase method and characterized by physicochemical tests. The nanoparticles had a mean particle size of 109 ± 5.4 nm and a Zeta potential of −38.7 ± 1.2 mV, the latter parameter indicating a stable formulation. These drug-loaded liposomes significantly decreased cell viability in vitro and tumor size in vivo, without generating systemic toxicity in the animals. There was evidence of cell cycle arrest and increased apoptosis. The promising results with the co-encapsulation of cisplatin/mifepristone warrant further research.

An investigation of targeted inhibition of transcription factor activity with pyrrole imidazole polyamide (PA) in chronic myeloid leukemia (CML) blast crisis cells.

Tyrosine kinase inhibitor (TKI) therapy is the standard treatment for chronic phase (CP)-chronic myeloid leukemia (CML), yet patients in blast crisis (BC) phase of CML are unlikely to respond to TKI therapy. The transcription factor E2F1 is a down-stream target of the tyrosine kinase BCR-ABL1 and is up-regulated in TKI-resistant leukemia stem cells (LSC). Pyrrole imidazole polyamides (PA) are minor groove binders which can be programmed to target DNA sequences in a gene-selective manner. This manuscript describes such an approach with a PA designed to down-regulate E2F1 controlled gene expression by targeting a DNA sequence within 100 base pairs (bp) upstream of the E2F1 consensus sequence. Human BC-CML KCL22 cells were assessed after treatment with PA, TKI or their combination. Our PA inhibited BC-CML cell expansion based on cell density analysis compared to an untreated control after a 48-hour time-course of PA treatment. However, no evidence of cell cycle arrest was observed among BC-CML cells treated with PA, with respect to their no drug control counterparts. Thus, this work demonstrates that PAs are effective in inhibiting E2F1 TF activity which results in a temporal reduction in BC-CML cell number. We envisage that PAs could be used in the future to map genes under E2F1 control in CML LSCs.

286PD - Post-treatment biopsies show evidence of cell cycle arrest and immune cell infiltration into tumors of ladiratuzumab vedotin-treated advanced breast cancer patients

286PD - Post-treatment biopsies show evidence of cell cycle arrest and immune cell infiltration into tumors of ladiratuzumab vedotin-treated advanced breast cancer patients

SCD \u2013 Stem Cell Differentiation Toward Osteoblast Onboard the International Space Station

Microgravity produces a variety of physical, chemical, and biological cues leading to an intricate and largely unresolved network of mechanosensitive molecules, transduction pathways, oxidative stress-related responses, and adaptations. The bone loss observed in astronauts and animal models after spaceflight is attributable to alterations in the bone tissue formation that depends on the continuous remodelling through the activities of bone-resorbing osteoclasts of hematopoietic lineage and bone-forming osteoblast of mesenchymal origin. Focusing on osteogenic differentiation, we present the results of the ”SCD - Stem Cells Differentiation” experiment, aiming to determine how human bone marrow stem cells (hBMSCs) react to a prolonged (approx. 2 weeks) exposure to microgravity in terms of growth, and differentiation when treated with a physiological osteo-inducer as 1,25-dihydroxy vitamin D (Vit D3). The experiment was selected by the European Space Agency and transferred to ISS with the Soyuz-TMA- 16M (ISS 42S). It was carefully prepared because experiments performed on ISS remain a uniquely exceptional means of clarifying the microgravitational effects on osteogenesis, often only partially activated and detectable under simulated conditions. Because of the substantial reduction in calcification observed (about 50% inflight vs. on-ground control), we looked at significantly affected pathways in hBMSCs grown in microgravity vs. on-ground controls. Genome-wide expression changes were assessed via microarray and next generation sequencing (NGS) and integrated with exosomal mi-RNA measurements. Multi-scale pathway analysis of the omics datasets revealed evidence of cell cycle arrest, occurring with a number of osteogenic gene markers, but without indications of adipogenesis, senescence and/or apoptosis.

Suppressive effects of sodium fluoride on cultured splenic lymphocyte proliferation in mice.

Fluoride-induced immunotoxicity has been documented in vivo, but limited reports have focused on the effects of fluoride on lymphocytes in vitro. Therefore, we have examined the suppressive effects of sodium fluoride on cultured splenic lymphocytes in mice. CD3+ T lymphocytes, CD19+ B lymphocytes, cytokines, and cell-cycle markers were analyzed through the use of a cell-counting kit, western blot, and flow cytometery. Splenic lymphocytes were isolated from 3-week-old male ICR mice and exposed to sodium fluoride (0, 100, 500, and 1000 μmol/L) for 24 h. The percentages of CD3+, CD3+CD4+, CD3+CD8+ T lymphocytes and CD19+ B lymphocytes were decreased (P<0.05 or P<0.01) in the sodium fluoride-exposed cells. This finding was correlated with the alterations in expression levels of cytokine proteins and with evidence of cell-cycle arrest. Thus, protein expression levels of IL-2, TNF-α, IFN-γ, TGF-β were decreased (P<0.05 or P<0.01), and IL-10 protein expression levels were increased (P<0.05 or P<0.01). The percentage of lymphocyte in G1 phase was significantly increased (P<0.05 or P<0.01), while expression levels of cyclin E/D and CDK2/4 were markedly decreased (P<0.05 or P<0.01). These findings demonstrate that sodium fluoride exposure suppresses splenic lymphocyte proliferation, which is represented by reducing populations and activation of splenic T and B lymphocytes. Alterations of cytokine protein expression and cell cycle arrest are the molecular basis of the sodium fluoride-suppressed splenic lymphocyte proliferation, while reduction of T lymphocytes and B lymphocytes is the explanation of sodium fluoride-decreased splenic immune function in vitro.

Transcriptional profiling of breast cancer cells in response to mevinolin: Evidence of cell cycle arrest, DNA degradation and apoptosis.

The merging of high-throughput gene expression techniques, such as microarray, in the screening of natural products as anticancer agents, is considered the optimal solution for gaining a better understanding of the intervention mechanism. Red yeast rice (RYR), a Chinese dietary product, contains a mixture of hypocholesterolemia agents such as statins. Typically, statins have this effect via the inhibition of HMG-CoA reductase, the key enzyme in the biosynthesis of cholesterol. Recently, statins have been shown to exhibit various beneficial antineoplastic properties through the disruption of tumor angiogenesis and metastatic processes. Mevinolin (MVN) is a member of statins and is abundantly present in RYR. Early experimental trials suggested that the mixed apoptotic/necrotic cell death pathway is activated in response to MVN exposure. In the current study, the cytotoxic profile of MVN was evaluated against MCF-7, a breast cancer-derived cell line. The obtained results indicated that MVN-induced cytotoxicity is multi-factorial involving several regulatory pathways in the cytotoxic effects of MVN on breast cancer cell lines. In addition, MVN-induced transcript abundance profiles inferred from microarrays showed significant changes in some key cell processes. The changes were predicted to induce cell cycle arrest and reactive oxygen species generation but inhibit DNA repair and cell proliferation. This MVN-mediated multi-factorial stress triggered specific programmed cell death (apoptosis) and DNA degradation responses in breast cancer cells. Taken together, the observed MVN-induced effects underscore the potential of this ubiquitous natural compound as a selective anticancer activity, with broad safety margins and low cost compared to benchmarked traditional synthetic chemotherapeutic agents. Additionally, the data support further pre-clinical and clinical evaluations of MVN as a novel strategy to combat breast cancer and overcome drug resistance.

Abstract A43: Evaluation of the cytotoxic effects of 3-O-acetyl-11-keto-β-boswellic acid in ovarian cancer cells

Abstract Ovarian cancer causes significant mortality, the five year survival rate is very low compared to other cancers, and most of the cases are diagnosed late, at stage III-IV. Most of the patients relapse and develop resistance to first line chemotherapy, therefore new strategies are urgently needed to improve survival and overcome resistance. Extracts from Boswellia sp. (Frankincense) used for centuries as a herbal medicine in Asia, are known to have anti-inflammatory effects and have more recently gained interest as potential anti-cancer agents. The active ingredients of Boswellia sp., boswellic acids, have a many effects on various cancer cells including induction of apoptosis, highlighting a putative role of boswellic acids as novel anti-cancer drugs, alone or in combination with other chemotherapies. This study examines the cytotoxic properties of the principal biologically active component of Frankincense, 3-O-acetyl-11-keto-β-boswellic acid (AKBA), in epithelial ovarian cancer cells, ultimately to evaluate its potential to subvert resistance to cisplatin. This study represents the first report of the potential utility of AKBA in the treatment of ovarian cancer. Using cisplatin-sensitive and resistant cell lines we have conducted a preliminary investigation into the cytotoxic mechanism of AKBA on ovarian cancer cells, focusing on DNA damage, the role of reactive oxygen species (ROS) and cellular responses, including induction of apoptosis and cell cycle perturbations. Methods: Ovarian cancer cell lines [UWB1.289 (BRCA1 mutant), A2780, and the cisplatin-resistant counterpart, A2780cis] were exposed to different concentrations of AKBA (5, 10, 15, 25 50 μM added as single dose) for different times (0, 6, 16, 24, 48 h). Flow cytometry was used to assess cell death and cell cycle status (propidium iodide staining) and apoptosis (propidium iodide/Annexin V staining). Analysis of DNA damage was examined using the alkaline comet assay. Further examinations of cell cycle perturbations using immunocytochemical analysis of α- and γ-tubulin; and ROS production using 2,7′-dichlorofluorescein diacetate and spectrofluorimetry are ongoing. Results: The data shows that 50 μM AKBA induced significant (P&amp;lt;0.001) DNA damage (strand breaks and alkali-labile sites) immediately in all cell lines compared to control exposure [0.5% v/v DMSO (AKBA solvent) or cell culture medium only], whereas 15 μM and 25 μM AKBA also induced significant DNA damage after longer than 16 h exposure. Significant cell death (PI staining; subG1/G0) was observed between 25 and 50 μM AKBA dependent on the exposure time in all cell lines, with UWB1.289 and A2780 showing greater cell death overall compared to A2780cis. These observations were confirmed by Annexin V staining, except that apoptosis was observed at the lower concentrations of AKBA (after 48 h exposure) compared to simple PI staining. Cell cycle analysis showed evidence of cell cycle arrest at G2/M in all cell lines following AKBA treatment, dependent on concentration and time of exposure. Conclusions: AKBA is cytotoxic to ovarian cancer cells, at pharmacologically realistic concentrations. AKBA also induces DNA damage and G2/M arrest, which may be related to its cytotoxic effects. AKBA may form the basis of a novel anticancer treatment for ovarian cancer perhaps alongside conventional chemotherapy. Citation Format: Kamla KSA AlSalmani, Marcus S. Cooke, Ikram A. Burney, Mark D. Evans. Evaluation of the cytotoxic effects of 3-O-acetyl-11-keto-β-boswellic acid in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A43.

Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.

Mallory–Denk bodies form when EZH2/H3K27me3 fails to methylate DNA in the nuclei of human and mice liver cells

EZH2/H3K27me3 and polycomb group complex (PcG) play a major role in regulating global gene expression including tumor suppressor genes. EZH2 is linked to cell cycle regulated EZH2 phosphorylation by CDK1, a mitotic kinase which increases in arrested mitosis compared to S phase. CDK1 phosphorylation of EZH2 accelerates the degradation of pEZH2. Phospho-EZH2 is subjected to ubiquitination. The half-like of pEZH2 is shorter when compared to total EZH2. In the present study, pEZH2 was found concentrated together with ubiquitin in the Mallory–Denk bodies (MDB) that were formed in hepatocytes in the livers of drug primed mice refed DDC and humans with alcoholic hepatitis or hepatocellular carcinoma. The cells that formed MDBs in the mice livers studied were associated with a growth advantage and a high proliferative index. However, the livers from patients with alcoholic hepatitis showed evidence of cell cycle arrest where PCNA, cyclin D1 and p27 positive nuclei were numerous but Ki-67 positive nuclei were scarce. It is concluded that MDB formation is linked to the cell cycle and global gene expression (i.e. loss of gene silencing) through its association with the regulation of the polycomb group PRC2/EZH2/H3K27me3 complex.

Abstract 4502: The small molecule TIC10 has potent anticancer efficacy mediated by induction of TRAIL production in normal and tumor cells

Abstract TRAIL is an attractive anti-neoplastic agent due to its minimal toxicity and its potent ability to induce apoptosis in cancer cells. Clinical trials with intravenous administration of recombinant human TRAIL in cancer patients have been limited by the short serum half-life of TRAIL and molecular resistance mechanisms including loss of cell surface death receptor expression and/or pro-survival expression changes in proteins that regulate apoptosis such as Mcl-1. Although TRAIL death receptor-targeted agonistic antibodies have an increased half-life, they act on one of the two pro-apoptotic TRAIL receptors DR4 (TRAIL-R1) or DR5 (TRAIL-R2). In order to pharmacologically increase tumor and host TRAIL production as a novel anti-neoplastic mechanism, to improve the pharmacokinetic properties of TRAIL by increasing its effective half-life, and perhaps its local concentration within the tumor microenvironment, we screened for small molecules capable of inducing the endogenous TRAIL gene. A cell-based bioluminescence reporter screen was conducted in TRAIL-resistant Bax-null human HCT116 colon cancer cells using the TRAIL gene promoter yielded TRAIL-inducing compound 10 (TIC10). TIC10 induces TRAIL at the message, membrane-bound, and soluble levels in a time- and dose-dependent manner in TRAIL-sensitive and TRAIL-resistant human cancer cell lines as well as normal human fibroblasts without any effect on cell cycle profile. TIC10 induces cell death in a number of cancer cell lines without evidence of cell cycle arrest, a characteristic of HDAC inhibitors that are the only other pharmacological agent currently shown to upregulate TRAIL. Lack of structural homology with HDAC inhibitors along with this observation suggest a novel mechanism of TRAIL-upregulation. Western blot analysis revealed that TIC10 sensitizes to TRAIL-mediated apoptosis by tumor cell-specific upregulation of DR5 and downregulation of Mcl-1. Human xenograft experiments demonstrate that TIC10 is highly effective as a multi- or single-dose agent with similar efficacy to TRAIL in TRAIL-sensitive xenografts. Some TRAIL-resistant xenografts such as RKO demonstrate TIC10 superiority to TRAIL with no incidence of toxicity. TIC10 causes highly increased protein levels of TRAIL and cleaved caspase-8, increased TUNEL staining indicating apoptosis, in addition to striking decrease in tumor cell density by histology in tumors of human xenografts in mice. Mice lacking tumors demonstrate an upregulation of TRAIL protein levels in serum. The effect of TIC10 on normal cell production of TRAIL provides a novel mechanism for killing tumor cells through a bystander effect in the tumor microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4502. doi:10.1158/1538-7445.AM2011-4502

Diversity of DNA damage response of astrocytes and glioblastoma cell lines with various p53 status to treatment with etoposide and temozolomide

Phosphorylation of histone H2AX is a sensitive marker of DNA damage, particularly of DNA double strand breaks. Using multiparameter cytometry we explored effects of etoposide and temozolomide (TMZ) on three glioblastoma cell lines with different p53 status (A172, T98G, YKG-1) and on normal human astrocytes (NHA) correlating the drug-induced phosphorylated H2AX (γH2AX) with cell cycle phase and induction of apoptosis. Etoposide induced γH2AX in all phases of the cell cycle in all three glioblastoma lines and led to an arrest of T98G and YKG-1 cells in S and G2/M. NHA cells were arrested in G1 with no evidence of γH2AX induction. A172 responded by rise in γH2AX throughout all phases of the cycle, arrest at the late S- to G2/M- phase, and appearance of senescence features: induction of p53, p21WAF1/CIP1, p16INK4A, and β-galactosidase, accompanied by morphological changes typical of senescence. T98G cells showed the presence of γH2AX in S phase with no evidence of cell cycle arrest. A modest degree of arrest in G1 was seen in YKG-1 cells with no rise in γH2AX. While frequency of apoptotic cells in all four TMZ-treated cell cultures was relatively low it is conceivable that the cells with extensive DNA damage were reproductively dead. The data show that neither the status of p53 (wild-type vs mutated, or inhibited by pifithrin-α) nor the expression of O6-methylguanine-DNA methyltransferase significantly affected the cell response to TMZ. Because of diversity in response to TMZ between individual glioblastoma lines our data suggest that with better understanding of the mechanisms, the treatment may have to be customized to individual patients.

Immunomodulatory Effects of the Triterpenoid CDDO after Allogeneic Bone Marrow Transplantation in Mice: Reduction of Acute Graft-Versus-Host Disease Lethality.

The use of hematopoietic stem cell transplantation (HSCT) in cancer treatment is seriously hampered by the occurrence of graft-versus-host disease (GVHD) and cancer relapse. During acute GVHD, inflammatory cytokines play a pivotal role in the amplification of GVHD. Therefore, assessment of agents with known anti-neoplastic activity that also reduce cytokine production may be useful in both the prevention of GVHD and cancer relapse. The synthetic triterpenoid, CDDO (2-cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid) is multifunctional molecule which has shown potent anti-cancer activities both in vitro and in vivo through the induction of apoptosis. We first examined the effects of CDDO on both human and murine T cell mitogen responses in vitro. CDDO significantly inhibited mitogen responsiveness of both human and murine T cells in vitro with evidence of cell cycle arrest of the human T cells. We then proceeded to examine the effects of CDDO on acute GVHD induction and progression. In these studies, lethally irradiated C57BL/6 mice received 10 million bone marrow cells (BMC) and 40 million spleen cells from fully MHC-mismatched BALB/c donors. All of the control mice succumbed rapidly due to acute GVHD. In contrast, the mice that received CDDO (120 ug/BID) given from days 0-3 following BMT exhibited significant improvement in survival (P < 0.001). Body weights from the treated mice also were significantly increased compared to untreated controls. We found that the timing of CDDO administration was a critical factor for protection from GVHD as protection only occurred when CDDO was administered early after BMT. Importantly, donor myeloid reconstitution was not adversely affected by CDDO treatment as determined by peripheral blood cell count and donor chimerism assessment on day +14 post-transplant. No adverse toxicities or effects on reconstitution were observed in the mice receiving BMC alone with CDDO being administered continuously. Given the reported direct anti-tumor effects of CDDO, it will be of particular interest in examining the effects of CDDO and allogeneic BMT in tumor-bearing recipients. Our findings suggest that CDDO can enhance the efficacy of allogeneic BMT by decreasing acute GVHD in mice.

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G0/G1 phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G0/G1 phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G0/G1 phase.

Early Induction of Apoptosis in Hematopoietic Cell Lines After Exposure to Flavopiridol

AbstractFlavopiridol (NSC 649890; Behringwerke L86-8275, Marburg, Germany), is a potent inhibitor of cyclin dependent kinases (CDKs) 1, 2, and 4. It has potent antiproliferative effects in vitro and is active in tumor models in vivo. While surveying the effect of flavopiridol on cell cycle progression in different cell types, we discovered that hematopoietic cell lines, including SUDHL4, SUDHL6 (B-cell lines), Jurkat, and MOLT4 (T-cell lines), and HL60 (myeloid), displayed notable sensitivity to flavopiridol-induced apoptosis. For example, after 100 nmol/L for 12 hours, SUDHL4 cells displayed a similar degree of DNA fragmentation to that shown by the apoptosis-resistant PC3 prostate carcinoma cells only after 3,000 nmol/L for 48 hours. After exposure to 1,000 nmol/L flavopiridol for 12 hours, typical apoptotic morphology was observed in SUDHL4 cells, but not in PC3 prostate carcinoma cells despite comparable potency (SUDHL4:120 nmol/L; PC3: 203 nmol/L) in causing growth inhibition by 50% (IC50). Flavopiridol did not induce topoisomerase I or II cleavable complex activity. A relation of p53, bcl2, or bax protein levels to apoptosis in SUDHL4 was not appreciated. While flavopiridol caused cell cycle arrest with decline in CDK1 activity in PC3 cells, apoptosis of SUDHL4 cells occurred without evidence of cell cycle arrest. These results suggest that antiproliferative activity of flavopiridol (manifest by cell cycle arrest) may be separated in different cell types from a capacity to induce apoptosis. Cells from hematopoietic neoplasms appear in this limited sample to be very susceptible to flavopiridol-induced apoptosis and therefore clinical trials in hematopoietic neoplasms should be of high priority.

Early Induction of Apoptosis in Hematopoietic Cell Lines After Exposure to Flavopiridol

Flavopiridol (NSC 649890; Behringwerke L86-8275, Marburg, Germany), is a potent inhibitor of cyclin dependent kinases (CDKs) 1, 2, and 4. It has potent antiproliferative effects in vitro and is active in tumor models in vivo. While surveying the effect of flavopiridol on cell cycle progression in different cell types, we discovered that hematopoietic cell lines, including SUDHL4, SUDHL6 (B-cell lines), Jurkat, and MOLT4 (T-cell lines), and HL60 (myeloid), displayed notable sensitivity to flavopiridol-induced apoptosis. For example, after 100 nmol/L for 12 hours, SUDHL4 cells displayed a similar degree of DNA fragmentation to that shown by the apoptosis-resistant PC3 prostate carcinoma cells only after 3,000 nmol/L for 48 hours. After exposure to 1,000 nmol/L flavopiridol for 12 hours, typical apoptotic morphology was observed in SUDHL4 cells, but not in PC3 prostate carcinoma cells despite comparable potency (SUDHL4:120 nmol/L; PC3: 203 nmol/L) in causing growth inhibition by 50% (IC50). Flavopiridol did not induce topoisomerase I or II cleavable complex activity. A relation of p53, bcl2, or bax protein levels to apoptosis in SUDHL4 was not appreciated. While flavopiridol caused cell cycle arrest with decline in CDK1 activity in PC3 cells, apoptosis of SUDHL4 cells occurred without evidence of cell cycle arrest. These results suggest that antiproliferative activity of flavopiridol (manifest by cell cycle arrest) may be separated in different cell types from a capacity to induce apoptosis. Cells from hematopoietic neoplasms appear in this limited sample to be very susceptible to flavopiridol-induced apoptosis and therefore clinical trials in hematopoietic neoplasms should be of high priority.

Intact G 2 -Phase Checkpoint in Cells of a Human Cell Line Lacking DNA-Dependent Protein Kinase Activity

Cells respond to radiation-induced DNA damage in a cell cycle phase-specific manner as shown by (1) variation in radiosensitivity across the cell cycle and (2) checkpoints in G1 and G2 phase at which arrest of progression of cells through the phases of the cell cycle occurs. We studied these processes in cells of human glioma cell lines which lack (M059J(PK-)) or express (M059K(PK+)) DNA-dependent protein kinase (DNA-PK) activity. Cell populations enriched with cells of a specific cell cycle phase were y-irradiated and analyzed for cell survival. Although both cell lines were relatively sensitive in G1 phase and resistant in S phase, the differential sensitivity was greater in M059J(PK-) cells. In the studies on checkpoints, unsynchronized cells were irradiated and examined for evidence of cell cycle arrest. Neither cell line showed a postirradiation G1-phase arrest, presumably because of mutant p53 status. For M059J(PK-) cells, all doses tested (2.5-10 Gy) resulted in a significant increase in the proportion of G2/M-phase cells; however, for M059K(PK+) cells, a significant increase in G2/M phase was observed only after 10 Gy. These results suggest that the ability to activate the G2-phase checkpoint remains intact in cells which lack DNA-PK activity.