RKO Colon Carcinoma Cells Research Articles

Evaluation of Anticancer Activity of Nucleoside-Nitric Oxide Photo-Donor Hybrids.

Herein, we report the synthesis of a new hybrid compound based on a 2'-deoxyuridine nucleoside conjugated with a NO photo-donor moiety (dU-t-NO) via CuAAC click chemistry. Hybrid dU-t-NO, as well as two previously reported 2'-deoxyadenosine based hybrids (dAdo-S-NO and dAdo-t-NO), were evaluated for their cytotoxic and cytostatic activities in selected cancer cell lines. dAdo-S-NO and dAdo-t-NO hybrids displayed higher activity with respect to dU-t-NO. All hybrids showed effective release of NO in the micromolar range. The photochemical behavior of the newly reported hybrid, dU-t-NO, was studied in the RKO colon carcinoma cell line, whereas the dAdo-t-NO hybrid was tested in both colon carcinoma RKO and hepatocarcinoma Hep 3B2.1-7 cell lines to evaluate the potential effect of NO released upon irradiation on cell viability. A customized irradiation apparatus for in vitro experiments was also designed.

Coumarin-modified ruthenium complexes: Synthesis, characterization, and antiproliferative activity against human cancer cells.

Among ruthenium complexes studied as anticancer metallodrugs, NKP-1339, NAMI-A, RM175, and RAPTA-C have already entered clinical trials due to their potent antitumor activity demonstrated in preclinical studies and reduced toxicity in comparison with platinum drugs. Considering the advantages of ruthenium-based anticancer drugs and the cytostatic activity of organometallic complexes with triazole- and coumarin-derived ligands, we set out to synthesize Ru(II) complexes of coumarin-1,2,3,-triazole hybrids (L) with the general formula [Ru(L)(p-cymene)(Cl)]ClO4. The molecular structure of the complex [Ru(2a)(p-cymene)(Cl)]ClO4 (2aRu) was determined by single-crystal X-ray diffraction, which confirmed the coordination of the ligand to the central ruthenium(II) cation by bidentate mode of coordination. Coordination with Ru(II) resulted in the enhancement of cytostatic activity in HepG2 hepatocellular carcinoma cells and PANC-1 pancreatic cancer cells. Coumarin derivative 2a positively regulated the expression and activity of c-Myc and NPM1 in RKO colon carcinoma cells, while the Ru(II) half-sandwich complex 2cRu induced downregulation of AKT and ERK signaling in PANC-1 cells concomitant with reduced intracellular levels of reactive oxygen species. Altogether, our findings indicated that coumarin-modified half-sandwich Ru(II) complexes held potential as anticancer agents against gastrointestinal malignancies.

Abstract 1553: miR-1 targets Cdk6 and controls cell cycle progression and apoptosis in colitis-associated dysplasia

Abstract Detection of colorectal dysplasia during surveillance colonoscopy is currently the best method of determining risk of colitis-associated colorectal cancer (CAC). An understanding of the early molecular changes associated with the development of these lesions will inform the identification of new biomarkers for earlier detection. miRNAs (miRs), highly conserved noncoding RNAs, show great promise as stable, tissue-specific biomarkers of neoplasia. We previously identified 12 miRs that are differentially-expressed in colitis-associated dysplasias (flat and polypoid) vs. inflamed colonic mucosa from mice treated with AOM/DSS. miR-1, a putative tumor suppressor, was downregulated in colitis-associated dysplasias. Analysis of the mRNA expression profile of AOM/DSS-induced dysplasias and prediction of the interactions between the miRs and their targets led to the selection of the Cdk6 as the target of miR-1 to be further investigated, based on its: 1) upregulation in AOM/DSS-induced dysplasia; and 2) association with cell cycle progression and inflammatory signaling. The goal of the present study was to validate the predicted interaction between miR-1 and Cdk6 and assess the biological function of miR-1 in vitro. The ability of miR-1 to interact with the 3’UTR of Cdk6 mRNA was assessed using a dual luciferase assay. Co-transfection of HCT116 or RKO colon carcinoma cells with Cdk6-WT and miR-1 mimics led to a significant reduction in relative luciferase activity in both cell lines (30%, p=0.0117 and 47%, p=0.0269; respectively). Transfection with the Cdk6-Mut did not alter relative luciferase activity, confirming the Cdk6 binding site was specific for miR-1. The biological function of miR-1 was assessed in HCT116 and RKO cells reverse-transfected with miR-1 and cel-miR-67 (negative control) for 48 hrs. Apoptosis (AnnexinV+ cells) and cell cycle progression (% of cells in G0/G1, G2/M and S phase) were evaluated by flow cytometry, and proliferation by cell count (Trypan Blue). HCT116 and RKO transfected with the miR-1 mimic exhibited a higher proportion of apoptotic cells than the negative control (30% and 20% increase, p=0.0022 and 0.0013, respectively). Cell cycle analyses revealed the miR-1 mimic induced cell cycle arrest (G0/G1) in both cell lines (p<0.05). In HCT116 cells, this was accompanied by a reduction in the percentage of cells in G2/M (p=0.002) and S (p<0.001) phase. No effect of miR-1 on total cell number was observed. These results demonstrate that Cdk6 is a direct target of miR-1, and suggest that downregulation of this miR in dysplastic lesions contributes to CAC by inducing cell cycle progression and inhibiting apoptosis. These data provide novel insight into the early molecular changes that accompany the development of colitis-associated dysplasia and may serve as biomarkers for early detection of neoplasia. Supported by the Timothy P. and Aurora M. Hughes Fund for Colon Cancer Research. Citation Format: Mariana F. Fragoso, Geysson J. Fernandez, Lisa Vanderveer, Harry S. Cooper, Michael Slifker, Margie L. Clapper. miR-1 targets Cdk6 and controls cell cycle progression and apoptosis in colitis-associated dysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1553.

In Preclinical Model of Ovarian Cancer, the SGK1 Inhibitor SI113 Counteracts the Development of Paclitaxel Resistance and Restores Drug Sensitivity

Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)–dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.

Herbal compound Teng-Long-Bu-Zhong-Tang inhibits metastasis in human RKO colon carcinoma.

Metastasis is one of the primary obstacles to the successful treatment of colorectal cancer. Teng-Long-Bu-Zhong-Tang (TLBZT) is a modern Chinese herbal formula that may be useful in the treatment of metastatic colorectal cancer. The present study evaluated the effects of TLBZT on lung metastasis in human RKO colon carcinoma cells injected into mice via the tail vein. The results demonstrated that TLBZT inhibited the metastasis of human RKO colon carcinoma cells to the lungs. TLBZT downregulated the expression of LOX and hypoxia-inducible factor 1α. TLBZT also inhibited the expression of integrins αV and β3 and the phosphorylation of focal adhesion kinase. These results indicate that TLBZT inhibits the lung metastasis of RKO colon carcinoma by regulating the expression of multiple genes. The results of the present study provide a new basis for the management of colorectal cancer metastasis using treatments derived from Chinese herbs.

Nicotine Modifies Cytokine Production by Human Mononuclears Stimulated by Colon Cancer Cells

Background: The undesirable effects of prolonged tobacco use and cigarette smoking are well-known and are due to inhalation of a great number of alkaloids including nicotine. Lung cancer is the most feared ailment in chronic smokers and its linkage with nicotine is not always justified. Notably, it has been shown that nicotine possesses anti-inflammatory properties and considering the link between chronic inflammation and cancer it is conceivable to assume that nicotine may reduce tumorigenesis. Our task was to examine the capacity of nicotine to promote peripheral blood mononuclear cells (PBMC) for cytokine production and to assess its ability to modify the cross-talk between immune and colon cancer cells. Methods: Unstimulated and LPS stimulated PBMC were incubated with various concentrations of nicotine and the generation of the cytokines TNFα, IL-1β, IL-6, IFNγ, IL-2, IL-10, and IL-1ra was detected. In addition, the production of these cytokines was examined in co-cultures of PBMC with HT-29 or RKO colon carcinoma cells in the presence of nicotine. Results: Nicotine caused inhibition of TNFα, IL-6, IL-1β and IL-1ra secretion by non-stimulated PBMC and reduced IL-6 and IFNγ production by mitogen stimulated cells. The effect of nicotine on the immune function of PBMC became prominent when the cells were triggered for cytokine production by HT-29 colon carcinoma cells. The secretion of the pro-inflammatory cytokines TNFα, IL-1β, IL-2 and IFNγ was inhibited, whereas that of IL-6 was not affected. Cytokine production by PBMC induced by RKO cells was not affected by nicotine at any of the concentrations used, suggesting cell-specific immune response. Conclusion: Nicotine inhibited the production of proinflammatory cytokines by PBMC. This effect was more conspicuous when PBMC were promoted by HT-29 colon carcinoma cells. It is feasible that nicotine, acting as an anti-inflammatory agent, may modulate the cross-talk between immune and cancer cells and to delay the progress of carcinogenesis.

Eugenol as a modulator of the immune dialogue between human mononuclears and colon cancer cells

Objective: Cloves (Syzygium aromaticum), the dried flower buds of a tree belonging to the Myrtaceae family have achieved a top place in herbal medicine. To its main active compound eugenol have been attributed definite anti-anaphylactic, anti-inflammatory, and even anti-cancer properties. It was the object of the study to elucidate the immunomodulatory potential of eugenol and its ability to alter the cross talk between human peripheral blood mononuclear cells (PBMC) and cells from two human colon carcinoma lines. Methods: Eugenol, at concentrations of 3.75, 7.5 and 15 �g/ml, was added for 24 hrs. to PBMC alone or to cells co-incubated with either HT-29 or RKO colon carcinoma cells. Subsequently, the generation of the following cytokines was examined: TNF-α, IL-1β, IL-6, IFN-γ, IL-1ra and IL-10. Results: Eugenol did not affect the capacity of unstimulated PBMC or that of cancer cells to produce any of the cytokines examined. However, when eugenol was added to PBMC co-incubated with HT-29 cells the production of all cytokines was inhibited, except for IL-6 and IFNγ. The capacity for cytokine generation by PBMC co-incubated with RKO cells was lowered concerning TNF-α and IL-1β. Conclusions: The results indicate that eugenol exerts an immunomodulatory effect on PBMC to produce inflammatory cytokines when stimulated by colon carcinoma cells from the two lines examined. This effect has been shown to be cancer cell type dependent. The capacity of eugenol to interfere with the cross-talk between immune and colon cancer cells may further explain the restraining effect of this compound on cancer development.

Abstract 2215: Pembrolizumab and ipilimumab reduce human RKO colon carcinoma tumor growth in HLA matched CD34-NSG humanized mice

Abstract Over the past decade there has been an increasing demand for preclinical models useful for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. The recently developed humanized mouse models composed of various types of human immune cells has the potential to recapitulate the human anti-tumor response modulated by checkpoint inhibitors. Tumor cells may develop multiple mechanisms to evade immune surveillance, including PD-L1 upregulation which suppresses T-cell effector responses towards the tumor. In the present study, we evaluated the efficacy of the checkpoint inhibitors pembrolizumab (anti-PD-1) and ipilimumab (anti-CTLA-4) in the human RKO colon carcinoma xenograft model in CD34-NSG humanized mice. These mice were engrafted with human hematopoietic stem cells (HSC) that matched the HLA haplotype of the RKO cells (HLA-A*01). Flow cytometry analysis confirmed high level of expression of PD-L1 in RKO colon carcinoma cells. The results from this study revealed that pembrolizumab and ipilimumab monotherapies significantly inhibited tumor growth. Surprisingly, combination therapy did not provide additive or synergistic effects and resulted in the same level of efficacy as the individual regimens. In order to understand the impact of these therapies on distinct immune cell populations, we analyzed the distribution of CD4 and CD8 T lymphocytes, Treg cells, NK cells and B cells in peripheral blood, spleen and tumor samples from tumor bearing humanized mice, via flow cytometry. We found that these checkpoint inhibitors can enhance effector functions of CD4+ and CD8+ T cells associated with increased expression of IFN-Γ. A parallel study with these checkpoint inhibitors carried out with humanized mice engrafted with HSC of the same haplotype as the RKO cells but from a different donor resulted in identical anti-tumor efficacy. In summary, these studies validate the use of humanized mouse models to test the tumor response to immune-checkpoint based therapies as it shows significant anti-tumor growth as a result of pembrolizumab and ipilimumab monotherapies associated with activation of T lymphocytes. Citation Format: Marcio Lasaro, Jason M. Davis, Mark Ellison, Martin R. Graf, Vivek Mahajan, Aidan Synnott, Robert J. Mullin, Paula L. Miliani De Marval. Pembrolizumab and ipilimumab reduce human RKO colon carcinoma tumor growth in HLA matched CD34-NSG humanized mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2215.

MiR-30e controls DNA damage-induced stress responses by modulating expression of the CDK inhibitor p21WAF1/CIP1 and caspase-3.

MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3′-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value.

In vitro toxicity of zinc oxide nanoparticles: a review

The toxic effect of ZnO nanoparticles is due to their solubility. ZnO nanoparticles dissolve in the extracellular region, which in turn increases the intracellular [Zn2+] level. The mechanism for increased intracellular [Zn2+] level and ZnO nanoparticles dissolution in the medium is still unclear. Cytotoxicity, increased oxidative stress, increased intracellular [Ca2+] level, decreased mitochondrial membrane potential, and interleukin-8 productions occur in the BEAS-2B bronchial epithelial cells and A549 alveolar adenocarcinoma cells following the exposure of ZnO nanoparticles. Confluent C2C12 cells are more resistant to ZnO nanoparticles compared to the sparse monolayer. Loss of 3T3-L1 cell viability, membrane leakage, and morphological changes occurs due to exposure of ZnO nanoparticles. ZnO nanoparticle induces cytotoxicity and mitochondrial dysfunction in RKO colon carcinoma cells. The occurrence of apoptosis, increased ROS level, reduced mitochondrial activity and formation of tubular intracellular structures are reported following exposure of ZnO nanoparticles in skin cells. Macrophages, monocytes, and dendritic cells are affected by ZnO nanoparticles. In addition, genotoxicity is also induced. The present review summarizes the literature on in vitro toxicity of ZnO nanoparticles (10–100 nm) on various cell lines.

Abstract 392: Helicase-like transcription factor (HLTF) expression and gene methylation in FFPE colon tumors

Abstract Background : HLTF, beside its roles in DNA binding, transcriptional and chromatin remodeling, is involved in post-replication DNA repair through its translocase and E3 ubiquitin ligase activities. HLTF is a tumor suppressor, and hypermethylation of its promoter was observed in 30-50% of gastrointestinal cancers. Moreover, HLTF inactivation in Apc-/+ mice induced the transition from colon adenocarcinoma to a carcinoma with high chromosomal instability. The epigenetic heterogeneity of the HLTF promoter could be related to the CpG island methylator phenotype of tumor suppressor genes in colorectal cancers. In the present study we started to investigate HLTF expression in colon cancer progression in relation to the epigenetic status of its gene promoter by using pyrosequencing. Methods : The HLTF promoter contains a 653 bp CpG island with 59 CpG sites (UCSC) among which 11 CpG sites were selected for this study. Genomic DNA was extracted from (a) HeLa (cervix adenocarcinoma) and RKO (colon carcinoma) cells and (b) from formalin-fixed paraffin-embedded (FFPE) colon tumors. DNA was treated with bisulfite and amplified by PCR the HLTF promoter fragment containing the 11 CpG sites. Pyrosequencing was performed to determine the relative methylation frequency at each site (HLTF PyroMark CpG Assay, Qiagen). HLTF expression was evaluated by immunohistochemistry (IHC) on tissue sections, and by RT-PCR and immunodetection on Western blot for the cell lines. Results : There was a negative correlation between promoter methylation and HLTF expression at the RNA and protein levels in RKO and HeLa cells. The average methylation rates were 92% for RKO and 25% for HeLa cells which presented HLTF repression and expression, respectively. IHC staining for HLTF was performed in 14 FFPE colon tumors and good pyrosequencing signals was obtained for DNA extracted from 6/14 samples. Three tumor groups were defined, based on the average methylation rate in the HLTF promoter : (a) < 30% ; (b) = 50% ; and (c) > 60%. In groups (a) and (b), the tissue was still well organized and HTLF protein expression was detected by IHC in the glandular epitelium. However, in group (c), the tumors were disorganized and HLTF protein expression was low or undetectable. These preliminary results suggest a correlation between 3 criteria : HLTF immunodetection, tissue organization and low methylation of the HLTF promoter. Two colon tumors did not express HLTF and presented a hypermethylated promoter. Conclusion : We could optimize pyrosequencing methylation analysis of genomic DNA extracted from 6 FFPE colon tumors, and confirmed that higher HLTF promoter methylation was associated with its repression in correlation with worse tumor phenotypes. This sensitive technique will now be applied to larger number of samples and additional CpG sites will be assessed. Citation Format: Ludovic Dhont, Alisson Di Stefano, Angela Szucs, Fadi Abdulsater, Isabelle Bar, Paul Delrée, Alexandra Belayew. Helicase-like transcription factor (HLTF) expression and gene methylation in FFPE colon tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 392. doi:10.1158/1538-7445.AM2014-392

Transcriptomes and shRNA suppressors in a TP53 allele-specific model of early-onset colon cancer in African Americans.

African Americans are disproportionately affected by early-onset, high-grade malignancies. A fraction of this cancer health disparity can be explained by genetic differences between individuals of African or European descent. Here the wild-type Pro/Pro genotype at the TP53Pro72Arg (P72R) polymorphism (SNP: rs1042522) is more frequent in African Americans with cancer than in African Americans without cancer (51% vs. 37%), and is associated with a significant increase in the rates of cancer diagnosis in African Americans. To test the hypothesis that Tp53 allele-specific gene expression may contribute to African American cancer disparities, TP53 hemizygous knockout variants were generated and characterized in the RKO colon carcinoma cell line, which is wild type for TP53 and heterozygous at the TP53Pro72Arg locus. Transcriptome profiling, using RNAseq, in response to the DNA-damaging agent etoposide revealed a large number of Tp53-regulated transcripts, but also a subset of transcripts that were TP53Pro72Arg allele specific. In addition, a shRNA-library suppressor screen for Tp53 allele-specific escape from Tp53-induced arrest was performed. Several novel RNAi suppressors of Tp53 were identified, one of which, PRDM1β (BLIMP-1), was confirmed to be an Arg-specific transcript. Prdm1β silences target genes by recruiting H3K9 trimethyl (H3K9me3) repressive chromatin marks, and is necessary for stem cell differentiation. These results reveal a novel model for African American cancer disparity, in which the TP53 codon 72 allele influences lifetime cancer risk by driving damaged cells to differentiation through an epigenetic mechanism involving gene silencing. TP53 P72R polymorphism significantly contributes to increased African American cancer disparity.

Sgk1 enhances RANBP1 transcript levels and decreases taxol sensitivity in RKO colon carcinoma cells

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.

A review of mammalian toxicity of ZnO nanoparticles

This review summarizes the literature on mammalian toxicity of ZnO nanoparticles (NPs) published between 2009 and 2011. The toxic effects of ZnO NPs are due to the compound's solubility. Whether the increased intracellular [Zn(2+)] is due to the NPs being taken up by cells or to NP dissolution in medium is still unclear. In vivo airway exposure poses an important hazard. Inhalation or instillation of the NPs results in lung inflammation and systemic toxicity. Reactive oxygen species (ROS) generation likely plays an important role in the inflammatory response. The NPs do not, or only to a minimal extent, cross the skin; this also holds for sunburned skin. Intraperitoneal administration induces neurological effects. The NPs show systemic distribution; target organs are liver, spleen, lung, and kidney and, in some cases, the heart. In vitro exposure of BEAS-2B bronchial epithelial cells and A549 alveolar adenocarcinoma cells results in cytotoxicity, increased oxidative stress, increased intracellular [Ca(2+)], decreased mitochondrial membrane potential, and interleukin (IL)-8 production. Decreased contractility of airway smooth muscle cells poses an additional hazard. In contrast to the results for BEAS-2B and A549 cells, in RKO colon carcinoma cells ZnO NPs and not Zn(2+) induce cytotoxicity and mitochondrial dysfunction. Short-term exposure of skin cells results in apoptosis but not in an inflammatory response, while long-term exposure leads to increased ROS generation, decreased mitochondrial activity, and formation of tubular intercellular structures. Macrophages, monocytes, and dendritic cells are affected; exposure results in cytotoxicity, oxidative stress, intracellular Ca(2+) flux, decreased mitochondrial membrane potential, and production of IL-1β and chemokine CXCL9. The NPs are phagocytosed by macrophages and dissolved in lysosomes. In vitro the Comet assay and the cytokinesis-blocked micronucleus assay show genotoxicity, whereas the Ames test does not. This is, however, not confirmed by in vivo genotoxicity assays. Protein binding results in increased stability.

The effect of p66shc protein on the resistance of the RKO colon cancer cell line to oxidative stress

P66shc protein is an alternative transcript product of the SHC1 gene. Whereas two other isoforms (p52shc and p46shc) have adaptor function in the RAS signaling pathway, p66shc regulates the amount of reactive oxygen species (ROS) in a cell. Previously, it was demonstrated that the p66shc genome knockout significantly extends maximal lifespan in mice. P66shc could translocate into the mitochondria and increase intracellular ROS level, although basic mechanisms of this activity remain poorly understood. P66shc seems to play a significant role in carcinogenesis since an increased expression of p66shc correlates with poor prognosis in colorectal carcinoma. In our study, we applied RNA interference using lentiviral constructions that express short hairpin RNA (shRNA) against the N-terminal CH2 domain of the p66shc isoform. As a consequence, the p66shc but not p52shc and p46shc isoforms was selectelively suppressed in the RKO colon carcinoma cell line. We found that RKO cells with p66shc knockdown have been shown to be more resistant to oxidative stress induced by hydrogen peroxide exposure or serum starvation. Moreover, mitochondrial fragmentation that depends on the mitochondrial ROS amount was significantly decreased in p66shc-deficient RKO cells. Our findings are consistent with the hypothesis that p66shc participates in mitochondrial accumulation of ROS during oxidative stress and consequently promotes induction of apoptosis.

The Effect of Radiation Quality on Genomic DNA Methylation Profiles in Irradiated Human Cell Lines

It has been acknowledged for many years that radiation exposure induces delayed, non-targeted effects in the progeny of the irradiated cell. Evidence is beginning to demonstrate that among these delayed effects of radiation are epigenetic aberrations, including altered DNA methylation. To test the hypothesis that differences in radiation quality affect radiation-induced DNA methylation profiles, normal AG01522 and RKO colon carcinoma cells were exposed to low-LET X rays and protons or high-LET iron ions. DNA methylation was then evaluated at delayed times using assays for p16 and MGMT promoter, LINE-1 and alu repeat element, and global methylation. The results of these experiments demonstrated radiation-induced changes in repeat element and global DNA methylation patterns at ∼20 population doublings postirradiation. Further, radiation-induced changes in repeat element and global DNA methylation were more similar between proton- and iron-ion-irradiated cells than X-irradiated cells, suggesting that radiation quality rather than LET alone affects the radiation-induced epigenetic profile. Since alterations in DNA methylation have also emerged as one of the most consistent molecular alterations in cancer, these data also suggest the possibility that radiation-induced carcinogenic risk might be affected by radiation quality.

Beryllium sulfate induces p21 CDKN1A expression and a senescence-like cell cycle arrest in susceptible cancer cell types

In fibroblasts, beryllium salt causes activation of the p53 transcription factor and induction of a senescence-like state. It is not known whether Be(2+) can affect the proliferation of cancer cells, which are generally unsusceptible to senescence. A172 glioblastoma and RKO colon carcinoma cell lines each have wildtype p53, so these cell types have the potential to be responsive to agents that activate p53. In A172 cells, BeSO(4) produced a G(0)/G(1)-phase cell cycle arrest and increased expression of senescence-associated β-galactosidase, an enzymatic marker of senescence. BeSO(4) caused phosphorylation of serine-15 of p53, accumulation of p53 protein, and expression of p21, the cyclin-dependent kinase inhibitor that is prominent during senescence. BeSO(4) inhibited A172 growth with an IC(50) = 4.7 μM in a 6-day proliferation assay. In contrast, BeSO(4) had no effect on RKO cells, even though Be(2+) uptake was similar for the two cell types. This differential responsiveness marks BeSO(4) as a reagent capable of activating a separable branch of the p53 signaling network. A172 and RKO cells are known to exhibit p53-dependent upregulation of p21 in response to DNA damage. The RKO cells produced high levels of p21 when exposed to DNA damaging agents, yet failed to express p21 when treated with BeSO(4). Conversely, BeSO(4) did not cause DNA damage in A172 cells, yet it was a potent inducer of p21 expression. These observations indicate that the growth control pathway affected by BeSO(4) is distinct from the DNA damage response pathway, even though both ultimately converge on p53 and p21.

Abstract 3151: Posttranscriptional regulation of p21 by MCG10, an RNA-binding protein and a target of tumor suppressor p53

Abstract The tumor suppressor p53 plays a crucial role in regulating many cellular processes, such as cell cycle arrest, apoptosis, senescence and cell metabolism, in response to stress signals. As a transcription factor, p53 modulates the expression of a plethora of genes, including p21, Killer/DR5, GADD45, TIGAR and MDM2. The tumor suppressor function of p53 is further underscored by the high propensity of p53 mutations found in human cancer. RNA-binding proteins are key modulators in RNA metabolism involved in alternative splicing, mRNA stability, mRNA transport as well as translation. Abnormal expression of RNA-binding proteins is implicated in a growing number of human diseases ranging from neurological disorders to cancer. Here, we showed that MCG10, an RNA-binding protein and a target of p53, regulates the stability of cell cycle inhibitor p21 transcript. Indeed, we found that MCG10 decreases p21 levels induced upon DNA damage. In addition, we showed that MCG10 directly binds to the 3′untranslated region of p21 transcript to destabilize it. Furthermore, we generated MCF7 breast adenocarcinoma and RKO colon carcinoma cell lines in which MCG10 is inducibly knocked down. We found that MCG10 is required for cell proliferation. In addition, we showed that deficiency in MCG10 leads to an increase in basal and DNA damage-induced p21 levels. Taken together, we uncovered a role for RNA-binding protein MCG10 in modulating the p53 pathway through posttranscriptional regulation of p21. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3151.

Selective Resistance of Tetraploid Cancer Cells against DNA Damage‐Induced Apoptosis

Aneuploidy and chromosomal instability, which are frequent in cancer, can result from the asymmetric division of tetraploid precursors. Genomic instability may favor the generation of more aggressive tumor cells with a reduced propensity for undergoing apoptosis. To assess the impact of tetraploidization on apoptosis regulation, we generated a series of stable tetraploid HCT116 and RKO colon carcinoma cell lines. When comparing diploid parental cells with tetraploid clones, we found that such cells were equally sensitive to a series of cytotoxic agents (staurosporine [STS], hydroxyurea, etoposide), as well as to the lysis by natural killer cells. In strict contrast, tetraploid cells were found to be relatively resistant against a series of DNA-damaging agents, namely cisplatin, oxaliplatin, camptothecin, and gamma- and UVC-irradiation. This increased resistance correlated with a reduced manifestation of apoptotic parameters (such as the dissipation of the mitochondrial transmembrane potential and the degradation of nuclear DNA) in tetraploid as compared to diploid cells subjected to DNA damage. Moreover, tetraploid cells manifested an enhanced baseline level of p53 activation. Inhibition of p53 abolished the difference in the susceptibility of diploid and tetraploid cancer cells to DNA damage-induced apoptosis. These data point to an intrinsic resistance of tetraploid cells against radiotherapy and DNA-targeted chemotherapy that may be linked to the status of the p53 system.

Analysis of dose-response effects on gene expression data with comparison of two microarray platforms

The problems of analyzing dose effects on gene expression are gaining attention in biomedical research. A specific challenge is to detect genes with expression levels that change according to dose levels in a non-random manner, but nonetheless may be considered as potential biomarkers. We are among the first to formally apply a tool that uses an isotonic (monotonic) regression approach to this area of study. We introduce a test statistic to select genes with significant dose-response expression in a monotonic fashion based on a permutation procedure. We then compare the results with those achieved from the application of a likelihood ratio-based test. We apply the isotonic regression approach to a study of gene expression in the RKO colon carcinoma cell line in response to varying dosage levels of the chemotherapeutic agent 5-fluorouracil. A feature of both Affymetrix and printed 75mer oligomer cDNA arrays produced from the same samples provides an opportunity to compare the two microarray platforms. Statistical software S-plus Code to implement the method is available from the authors. kcoombes@mdanderson.org