Majority Of Cancer Cells Research Articles

Telomerase Cajal body protein 1 depletion inhibits telomerase trafficking to telomeres and induces G1 cell cycle arrest in A549 cells.

Telomerase Cajal body protein 1 (TCAB1) is a telomerase holoenzyme, which is markedly enriched in Cajal bodies (CBs) and facilitates the recruitment of telomerase to CBs in the S phase of the cell cycle. This recruitment is dependent on TCAB1 binding to a telomerase RNA component. The majority of cancer cells are able to grow indefinitely due to telomerase and its mechanism of trafficking to telomeres. In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes. In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death. Overall, these observations indicated that TCAB1 may be essential for A549 cell proliferation and cell cycle regulation, and may be a potential candidate for the development of a therapeutic target for lung adenocarcinomas.

RB in glutamine metabolism.

RB in glutamine metabolism.

The inhibitory effect of helenalin on telomerase activity is attributed to the alkylation of the CYS445 residue: Evidence from QM/MM simulations

Enhanced telomerase activity is a hallmark in the majority of cancer cells. Thus, understanding the interactions between telomerase and its inhibitors is fundamentally important for the development of novel anticancer drugs without severe side effects. In this study, the covalent binding of helenalin to CYS445 of telomerase (PDB ID: 3DU6) was simulated using combined quantum chemical and molecular mechanical (QM/MM) methods. The results showed that the reaction was a reversible Michael-type addition and a hydrogen bond was formed between helenalin and the side chain of LYS416 of telomerase during the reaction procedure. The LYS416 residue is vital to telomere DNA recognition by interacting with DNA base through hydrogen bonds. The alkylation of CYS445 of telomerase by helenalin may interfere with the telomere DNA recognition at the telomerase active site, thus resulting in inhibition of the enzyme activity.

Effect of atmospheric gas plasmas on cancer cell signaling

Cancer is one of the most life-threatening diseases with many forms still regarded as incurable. The conventional cancer treatments have unwanted side effects such as the death of normal cells. A therapy that can accurately target and effectively kill tumor cells could address the inadequacies of the available therapies. Atmospheric gas plasmas (AGP) that are able to specifically kill cancerous cells offer a promising alternative approach compared to conventional therapies. AGP have been shown to exploit tumor-specific genetic defects and a recent trial in mice has confirmed its antitumor effects. The mechanism by which the AGP act on tumor cells but not normal cells is not fully understood. A review of the current literature suggests that reactive oxygen species (ROS) generated by AGP induce death of cancer cells by impairing the function of intracellular regulatory factors. The majority of cancer cells are defective in tumor suppressors that interfere normal cell growth pathways. It appears that pro-oncogene or tumor suppressor-dependent regulation of antioxidant/or ROS signaling pathways may be involved in AGP-induced cancer cell death. The toxic effects of ROS are mitigated by normal cells by adjustment of their metabolic pathways. On the other hand, tumor cells are mostly defective in several regulatory signaling pathways which lead to the loss of metabolic balance within the cells and consequently, the regulation of cell growth. This review article evaluates the impact of AGP on the activation of cellular signaling and its importance for exploring mechanisms for safe and efficient anticancer therapies.

Toward the design of new DNA G-quadruplex ligands through rational analysis of polymorphism and binding data

Human telomeres play a key role in protecting chromosomal ends from fusion events; they are composed of d(TTAGGG) repeats, ranging in size from 3 to 15 kb. They form G-quadruplex DNA structures, stabilized by G-quartets in the presence of cations, and are involved in several biological processes. In particular, a telomere maintenance mechanism is provided by a specialized enzyme called telomerase, a reverse transcriptase able to add multiple copies of the 5′-GGTTAG-3′ motif to the end of the G-strand of the telomere and which is over-expressed in the majority of cancer cells. The central cation has a crucial role in maintaining the stability of the structure. Based on its nature, it can be associated with different topological telomeric quadruplexes, which depend also on the orientation of the DNA strands and the syn/anti conformation of the guanines. Such a polymorphism, confirmed by the different structures deposited in the Protein Data Bank (PDB), prompted us to apply a computational protocol in order to investigate the conformational properties of a set of known G-quadruplex ligands and their molecular recognition against six different experimental models of the human telomeric sequence d[AG3(T2AG3)3]. The average AutoDock correlation between theoretical and experimental data yielded an r2 value equal to 0.882 among all the studied models. Such a result was always improved with respect to those of the single folds, with the exception of the parallel structure (r2 equal to 0.886), thus suggesting a key role of this G4 conformation in the stacking interaction network. Among the studied binders, a trisubstituted acridine and a dibenzophenanthroline derivative were well recognized by the parallel and the mixed G-quadruplex structures, allowing the identification of specific key contacts with DNA and the further design of more potent or target specific G-quadruplex ligands.

Abstract 4055: Non-canonical telomere maintenance mechanism in B-cell chronic lymphocytic leukemia.

Abstract Unlimited cancer cells proliferation requires mechanisms that counteract telomere attrition. In the majority of cancer cells this is possible through up-regulation of telomerase activity. Alternatively, cancer cells use homologous recombination between telomeres. This second mechanism called ALT (Alternative Lengthening of Telomere) implicates proteins that either elongates telomeres or that either prevents telomere loss (maintenance). Among these proteins a complex composed of the topoisomerase III alpha (hTopoIIIα), BLM, RMI1 and 2 is require to repair stalled replication forks. Little is known about the involvement of the ALT mechanism in B-cell chronic lymphocytic leukemia (B-CLL), which shows low telomerase activity, shelterin defect and telomeric dysfunctions. We found that hTopoIIIα gene expression is downregulated in almost all B-CLL patients tested (94%; n=31), with 60% of B-CLL patients presenting a two time decreased compared to control samples. That prompted us to build the first CpG islands map of the hTopoIIIα promoter and to characterize those that are methylated in B-CLL patients. To investigate the methylation impact, we first treated CLL cell lines with 5-Azacytidine, a chemical analogue of cytidine that induces hypomethylation, and demonstrated that this treatment increases hTopoIIIα expression. In line, luciferase experiments revealed that SSSI-induced CpG islands methylation inside the TopoIIIα promoter leads to a transcriptional inhibition. Nevertheless, deletion mutants of the TopoIIIα promoter suggested that CpG islands found to be methylated in B-CLL patients are not implicated in TopoIIIα expression regulation through methylation. Interestingly, we didn't observe a good correlation between mRNA and protein expressions in all B-CLL patients (n=6). Indeed some patients with a moderate amount of mRNA showed low level of the corresponding protein hTopoIIIα (<50% compared to controls; n=3). Downregulation of hTopoIIIα may increase recombination rate between sister chromatid. Hence, experiments aiming to evaluate sister chromatid exchange and telomeric sister chromatid exchange rate on B-CLL patients samples according to their hTopoIIIα expression level have been performed and the data will be discuss. Our results suggest that there is no canonical mechanism maintaining telomeres in B-CLL but rather an overall recombination arising during replication, due to hTopoIIIα downregulation, leading to a silenced genetic instability in the early step of the disease. Citation Format: Sandrine Medves, Morgan Auchter, Laetitia Chambeau, Sophie Gazzo, Aurélie Verney, Etienne Moussay, Wim Ammerlaan, Hamid Morjani, Vincent Géli, Valérie Palissot, Gilles Salles, Guy Berchem, Thomas Wenner. Non-canonical telomere maintenance mechanism in B-cell chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4055. doi:10.1158/1538-7445.AM2013-4055

Abstract 3417: Increased anticancer effect of combining Wee1 (MK1775) and Chk1 (AZD7762) inhibitors in malignant melanoma cell lines.

Abstract Malignant melanoma is one of the most increasing forms of cancer worldwide, in addition to being notoriously resistant to therapy. Despite recent advances, there are no curative treatments once the cancer has spread (stage IV); there is thus a precarious need to find new and better treatment alternatives. Unlike normal cells, the majority of cancer cells have a defective G1/S DNA-damage checkpoint and are therefore more dependent on the G2/M checkpoint when DNA damages arise. Central in regulating the latter checkpoint is Wee1, a kinase which may stop the cell cycle by adding a negative phosphorylation on CDK1. Wee1 is regulated by Chk1 which upon DNA damage can also inhibit the complimentary counterpart of Wee1; CDC25. In recent studies, Wee1 and Chk1 have additionally been indicated as key players in regulating DNA replication in S-phase. We have previously reported that increased Wee1 protein expression was associated with malignancy in melanoma tumour samples, and that siWee1 transfection led to reduced viability in melanoma cell lines. In the present study we have combined inhibitors of Wee1 (MK1775) and Chk1 (AZD7762) in a panel consisting of both metastatic melanoma- and normal cells, including a Plexxikon resistant melanoma cell line (MelJR post3.3). We found that the combination synergistically reduced the amount of viable cells in the melanoma cell lines (∼50-80% reduction), whilst only having very moderate effect (∼10% reduction) in the normal cells. Interestingly, the reduction in number of viable cells was found in both p53WT and p53Mut cell lines, indicating that the p53 status do not predict the response to such treatment. Furthermore, combinational treatment with the inhibitors caused increased cleavage of PARP and caspases, indicative of apoptosis, as compared to MK1775 or AZD7762 treatment alone. The combination also caused a marked increase in DNA double-strand breaks, as demonstrated by augmented levels of γ-H2AX (predominantly found in S-phase cells). In line with this, preliminary results from melanoma cells grown as spheroids, revealed the same tendency, with approximately 50% reduction in spheroid volume when combining the inhibitors. In vivo experiments using melanoma cells grown in SCID mice are currently ongoing. In conclusion, our results indicate a potential new role for inhibitors of Wee1 and Chk1 in treatment of malignant melanoma. Citation Format: Gry I. Magnussen, Vivi A. Florenes, Birgit Engesæter, Elisabeth Emilsen. Increased anticancer effect of combining Wee1 (MK1775) and Chk1 (AZD7762) inhibitors in malignant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2013-3417

Abstract 2455: BAM38 is a potent and selective pro-apoptotic BAX activator molecule that restores apoptosis in cancer cells.

Abstract Targeting key apoptosis regulators to overcome the apoptotic resistance of cancer cells is a highly attractive therapeutic strategy. The BCL-2 protein family includes both pro- and anti-apoptotic proteins that form a complex interaction network and regulate the critical balance between cellular life and death. BAX is a pro-apoptotic BCL-2 member that, when activated, undergoes a structural transformation, which converts BAX from an inactive conformation into a lethal one, creating mitochondrial pores. Although inactivating mutations in BAX have been identified with a very small frequency in tumors, cancer cells commonly ensure survival by overexpression of anti-apoptotic BCL-2 members, a feature that contributes to tumorigenesis and chemoresistance. Therefore, the vast majority of cancer cells contain functional but suppressed BAX. Our discovery of the BAX trigger site and BAX activator molecule 7 (BAM7), which directly triggers BAX activation and selective BAX-mediated cell death, provides a new opportunity for rational drug design to target BAX and promote cell death in tumors. Using an in silico structural similarity approach and a competitive fluorescence polarization assay, we identified a novel small molecule activator of BAX, BAM38, that has ten-fold improved binding potency compared to BAM7 and retains selectivity over anti-apoptotic BCL-2 proteins. NMR and molecular docking analysis demonstrates that BAM38 engages the BAX trigger site using increased complementary interactions. Biochemical assays indicate that BAM38 has correspondingly improved potency in triggering BAX conformational activation and BAX-mediated membrane permeabilization. In a panel of acute myeloid leukemia (AML) cells that overexpress distinct combinations of anti-apoptotic BCL-2 family proteins, BAM38 overcomes apoptotic blockade through induction of BAX-mediated apoptosis. We observe dose-dependent AML cell death that coincides with BAX translocation, cytochrome c release and caspase 3/7 activation. BAM38 synergizes with the BCL-2/BCL-XL inhibitor ABT-737 in resistant AML cells, highlighting the therapeutic potential of combined direct pharmacologic activation of BAX with targeted inhibition of anti-apoptotic BCL-2 and BCL-XL. Thus, our data suggest a novel small molecule approach to restoring cancer cell death by directly targeting BAX, and provides a lead molecule for further development into a next generation cancer therapeutic. Citation Format: Denis E. Reyna, Loren D. Walensky, Evripidis Gavathiotis. BAM38 is a potent and selective pro-apoptotic BAX activator molecule that restores apoptosis in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2455. doi:10.1158/1538-7445.AM2013-2455 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2471: Chemical optimization of direct and selective molecular activators of pro-apoptotic BAX for cancer therapy.

Abstract BAX is a pro-apoptotic BCL-2 family member that lies dormant in the cytosol until triggered by cellular stress to translocate to the mitochondria and form toxic oligomeric pores. The majority of cancer cells retain wild-type BAX and instead neutralize the death pathway by overexpressing anti-apoptotic BCL-2 family proteins. Thus, direct activation of BAX represents a pharmacologic opportunity to lower the apoptotic threshold in the setting of chemoresistance. Using a Stabilized Alpha-Helix of BCL-2 domain (SAHB) modeled after the BIM BH3 helix, we previously identified the “trigger site” for direct BAX activation at the confluence of alpha-helices 1 and 6. In silico screening for small molecule modulators of BAX led to our identification of BAX activator molecule 7 (BAM7), which selectively engages the trigger site and promotes BAX-mediated cell death (Gavathiotis et al, Nat Chem Biol, 2012). Here, we applied BAM7 to a panel of acute lymphoblastic leukemia cells and observe dose-responsive killing in the micromolar range. To improve the potency of anti-ALL activity, we undertook a systematic medicinal chemistry-based iteration of BAM7’s molecular features. A series of analogs demonstrate improved BAX binding activity based on a FITC-BIM SAHB/BAX competitive fluorescence polarization assay. The most potent binders were advanced to testing in a cellular model of anti-apoptotic protein-dependent chemoresistance. Whereas select BAM7 derivatives exhibited a potency similar to or greater than the BCL-2/BCL-XL inhibitor ABT-737 in a BCL-XL-dependent ALL cell line, the compounds significantly outperformed ABT-737 in the isogenic MCL-1-dependent ALL cells. Preliminary studies revealed favorable pharmacokinetic profiles for lead BAM7 derivatives, including oral bioavailability and central nervous system penetration. These early encouraging structure-activity relationship and pharmacokinetic data on chemically-optimized BAM7 analogs suggest that direct and selective activation of BAX may be a viable strategy for therapeutic induction of apoptosis in the context of cancer chemoresistance. Citation Format: Joseph A. Bellairs, Denis Reyna, Divakaramenon Sethumadhavan, Madhavi Neelagiri, Alex Broadhead, Christopher Baccei, Brian Koss, Joseph T. Opferman, Evripidis Gavathiotis, Loren D. Walensky. Chemical optimization of direct and selective molecular activators of pro-apoptotic BAX for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2013-2471

Pharmacophore modeling and 3D-QSAR (CoMSIA) studies for structural requirements of some triazine derivatives as G-quadruplex binders for telomerase inhibition

Human telomeres are repetitive guanine rich sequences found at the ends of chromosomes that form special secondary structures known as G-quadruplexes. In majority of cancer cells, the telomerase enzyme has been found to maintain the length of telomeres, conferring cell immortality and prevention of cell senescence. Small molecules that bind and stabilize the G-quadruplex have proved to be potential telomerase inhibitors. The quantitative structure–activity relationships of triazine derivatives as G-quadruplex mediating telomerase inhibitors remain unclear. The objective of the study is to uncover structural requirements for triazine derivatives as G-quadruplex binders, which would eventually assist and complement the rational drug-design attempts for successful development of telomerase inhibitors as anticancer drugs. The model has been established by using a training set of compounds with cross-validated q2 0.593, non-cross-validated r2 0.976, standard error of estimate 0.167, and F test 115.130 for the best CoMSIA model. The predictive ability of the models was validated with the correlation coefficient rpred2 0.738 for the best CoMSIA model. The highly predictive 3D-model resulting from this study discovers the roles played by molecular features, mainly steric, hydrogen bond donor and hydrophobic properties.

Molecular Recognition of Human Telomeric DNA by Phenanthroline-Based G-Quadruplex Ligands

G-quadruplexes (G4) are non-canonical DNA structures assumed by guanine rich sequences. G4 are stabilized by the presence of cations and are characterized by a high degree of structural polymorphism with different patterns of groove, loop arrangement, strand orientations and stoichiometry. G-rich sequences are over-represented in the promoter regions of many oncogenes as well as at human telomeres, d(TTAGGG) repeats, ranging in size from 3 to 15 kb, involved in protecting chromosomal ends. A specialized enzyme, called telomerase, provides a telomere maintenance mechanism by elongating the end of the G-strand and it is activated in the majority of cancer cells. Therefore there are two general strategies of telomerase targeting in cancer treatment. One is a direct targeting of telomerase to cause its inhibition; the other one is the use of G4 stabilizers which block telomerase access to telomere, thus causing an indirect enzyme inhibition. Here, we evaluated the molecular recognition of some phenanthroline-based ligands against four different experimental models of the human telomeric sequence d[AG3(T2AG3)3] by means of docking simulations. Our theoretical analysis was able to reproduce the experimental affinity measurements, with a linear squared correlation factor r2 equal to 0.719 among all the studied models. These findings highlighted the importance to consider the polymorphism of the DNA G4. Interestingly, this correlation resulted always improved with respect to that of the single folds, with the exception of the parallel structure, thus suggesting a key role of this G4 conformation in the interaction network of the tested binders. Moreover, we identified the moieties of the phenanthroline scaffold directly involved in the complex formation. This allowed to rationalize the improved binding affinity always associated with a bis-phenanthroline system and to explain why a phenanthroline substituted with a pyridine ring is favored with respect to the pyrimidine one.

Evaluation of Urinary Human Telomerase Reverse Transcriptase mRNA and Scatter Factor Protein as Urine Markers for Diagnosis of Bladder Cancer

Expression of the human telomerase reverse transcriptase (hTERT) gene, which codes for the catalytic subunit of telomerase is considered an important tumor marker used for bladder cancer detection being found in the majority of cancer cells. Scatter Factor (SF) is a secretory protein produced by fibroblasts and smooth muscles and induces scattering of the epithelial cells. The aim of the current study was to evaluate the potential usefulness of hTERT and SF measurement as urinary markers for bladder cancer diagnosis. Voided urine specimens were collected from patients with histologically confirmed bladder urothelial carcinoma (malignant group: n = 60), urological patients without urothelial carcinoma (benign group: n = 25), and healthy volunteers (control group: n = 20). All cases underwent urine cytology, serological schistosomiasis antibody assay and detection of urinary hTERT mRNA using RT-PCR and SF using ELISA. Positivity rate of hTERT mRNA was markedly higher in malignant versus benign or control cases (86.67%, 8%, and 0%, respectively, p-value < 0.001). Combining hTERT and cytology increased the sensitivity of cytology to 95%. According to a cut-off value of urinary SF (> or = 410 ng/mg protein), 57 (95%) of the patients with bladder carcinoma, 10 (40%) with benign lesions, and non of the control individuals were positive and the difference between the 3 groups was statistically significant (p < 0.001). The sensitivity of cytology was increased to 98.33% when combined with the SF assay. When associating the two urinary markers with different clinicopathological factors of the bladder cancer group, only SF exerted a significantly higher positivity rate at the invasive stage (100%) than the superficial stage (88.46%) as well as in transitional cell carcinoma (100% thansquamous cell carcinoma type (87.5%). hTERT and SF can be considered potential useful markers for detection of bladder cancer.

Abstract P2-06-05: Single-cell RNA sequencing of paclitaxol-treated breast cancer cell lines to find individual cell response

Abstract Cancer treatments act on a population of cells, each of which may experience different individual responses to treatment. Such differential response will result in resistance to treatment even if a majority of cancerous cells are eliminated. To examine differential cell response, we simultaneously profiled the gene expression and mutation spectrum of individual cells from the MDAMB231 cell line using next generation sequencing of isolated RNA. A total of 23 transcriptomes were characterized from paclitaxel-treated and paclitaxel-surviving cells. We found significant different changes in mutation rates between paclitaxel treated cells, with a dose-dependent increase in single nucleotide changes in RNA in paclitaxel-treated cells. Cells undergoing exposure to paclitaxel also showed higher pathway activity in SRC, as well as an integrin switch from ITGB1 to ITGB3. In contrast, cells that survived a high dose of paclitaxel showed an insignificant number of single nucleotide changes, suggesting that these cells either evaded initial paclitaxel exposure or were better able to repair the effects of paclitaxel exposure. Despite the RNA sequence similarity between surviving and untreated cells, there were changes in gene expression and pathway activities including higher PI3K activity. Paclitaxel-surviving cells also showed activation of pathways associated with higher proliferation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-06-05.

Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy

Nearly a century ago, cell biologists postulated that the chromosomal aberrations blighting cancer cells might be caused by a mysterious organelle-the centrosome-that had only just been discovered. For years, however, this enigmatic structure was neglected in oncologic investigations and has only recently reemerged as a key suspect in tumorigenesis. A majority of cancer cells, unlike healthy cells, possess an amplified centrosome complement, which they manage to coalesce neatly at two spindle poles during mitosis. This clustering mechanism permits the cell to form a pseudo-bipolar mitotic spindle for segregation of sister chromatids. On rare occasions this mechanism fails, resulting in declustered centrosomes and the assembly of a multipolar spindle. Spindle multipolarity consigns the cell to an almost certain fate of mitotic arrest or death. The catastrophic nature of multipolarity has attracted efforts to develop drugs that can induce declustering in cancer cells. Such chemotherapeutics would theoretically spare healthy cells, whose normal centrosome complement should preclude multipolar spindle formation. In search of the 'Holy Grail' of nontoxic, cancer cell-selective, and superiorly efficacious chemotherapy, research is underway to elucidate the underpinnings of centrosome clustering mechanisms. Here, we detail the progress made towards that end, highlighting seminal work and suggesting directions for future research, aimed at demystifying this riddling cellular tactic and exploiting it for chemotherapeutic purposes. We also propose a model to highlight the integral role of microtubule dynamicity and the delicate balance of forces on which cancer cells rely for effective centrosome clustering. Finally, we provide insights regarding how perturbation of this balance may pave an inroad for inducing lethal centrosome dispersal and death selectively in cancer cells.

Novel regulation of checkpoint kinase 1: Is checkpoint kinase 1 a good candidate for anti‐cancer therapy?

DNA-damaging strategies, such as radiotherapy and the majority of chemotherapeutic therapies, are the most frequently used non-surgical anti-cancer therapies for human cancers. These therapies activate DNA damage/replication checkpoints, which induce cell-cycle arrest to provide the time needed to repair DNA damage. Due to genetic defect(s) in the ATM (ataxia-telangiectasia mutated)-Chk2-p53 pathway, an ATR (ATM- and Rad3-related)-Chk1-Cdc25 route is the sole checkpoint pathway in a majority of cancer cells. Chk1 inhibitors are expected to selectively induce the mitotic cell death (mitotic catastrophe) of cancer cells. However, recent new findings have pointed out that Chk1 is essential for the maintenance of genome integrity even during unperturbed cell-cycle progression, which is controlled by a variety of protein kinases. These observations have raised concerns about a possible risk of Chk1 inhibitors on the clinics. In this review, we summarize recent advances in Chk1 regulation by phosphorylation, and discuss Chk1 as a molecular target for cancer therapeutics.

CANCEROUS IMMUNOGLOBULINS AND CA215: IMPLICATIONS IN CANCER IMMUNOLOGY

Immunoglobulins are typically expressed by B cells in our normal immune system. However, certain normal human tissues, such as hyperplastic epithelial cells, cells of the immunologically privileged sites and the majority of cancer cells, have also been found to be sites of immunoglobulin production. Current research is lacking in regards to the differential immunoglobulin expression, the underling mechanisms of action and the biological implications of these cancerous immunoglobulins in cancer immunology. This article reviews the etiology of atypical immunoglobulin expression in normal non-B cells and cancer cells, with emphasis on the exploration of the possible mechanisms of action and biological function of these atypical immunoglobulins, by means of specific biological probes. In contrast to immunoglobulins of B cell origins, atypical immunoglobulins were found to carry additional post-translational modifications, including a unique carbohydrateassociated epitope recognized by RP215 monoclonal antibody. This unique RP215-specific epitope enables us to differentiate between these two types of immunoglobulins. Atypical immunoglobulins expressed by cancer cells have been a common subject of interest in cancer immunology. Furthermore, the recent accumulation of experimental evidence has indicated that these atypical immunoglobulins are essential for the growth and proliferation of cancer cells under our normal immune environment. RP215 monoclonal antibody also reacts with many other cancer cell-expressed glycoproteins, known as CA215, on the cancer cell surface. Apoptosis of cultured cancer cells can be induced and growth inhibition of implanted tumors can be observed in nude mouse animal models. Therefore, humanized RP215 monoclonal antibody, which reacts mainly with surface bound CA215, may have the potential to be developed as an anti-cancer drug for the treatment of human cancers. A better understanding of cancer cell-expressed immunoglobulins not only improves our knowledge of cancer immunology, but also benefits cancer patients in the field of cancer monitoring and therapeutic treatments.

Ortho-Quinone tanshinones directly inhibit telomerase through an oxidative mechanism mediated by hydrogen peroxide.

The tanshinone natural products possess a variety of pharmacological properties including anti-bacterial, anti-inflammatory, anti-oxidant, and anti-neoplastic activity. The molecular basis of these effects, however, remains largely unknown. In the present study, we explored the direct effect of tanshinones on the enzyme telomerase. Telomerase is up-regulated in the majority of cancer cells and is essential for their survival, making it a potential anti-cancer drug target. We found that the ortho-quinone tanshinone II-A inhibits telomerase in a time- and DTT-dependent fashion, and the hydrogen peroxide scavenger catalase protected telomerase from inactivation. These findings demonstrate that ortho-quinone containing tanshinones can inhibit telomerase owing to their ability to generate reactive oxygen species. The results also provide evidence that telomerase is directly and negatively regulated by reactive oxygen species.

M2 Macrophages Induced by Prostaglandin E2 and IL-6 from Cervical Carcinoma Are Switched to Activated M1 Macrophages by CD4+ Th1 Cells

Monocytes attracted by tumor-induced chronic inflammation differentiate to APCs, the type of which depends on cues in the local tumor milieu. In this work, we studied the influence of human cervical cancer cells on monocyte differentiation and showed that the majority of cancer cells either hampered monocyte to dendritic cell differentiation or skewed their differentiation toward M2-like macrophages. Blocking studies revealed that M2 differentiation was caused by tumor-produced PGE(2) and IL-6. TGF-β, IL-10, VEGF, and macrophage colony-stimulating factor did not play a role. Notably, these CD14(+)CD163(+) M2 macrophages were also detected in situ. Activation of cancer cell-induced M2-like macrophages by several TLR-agonists revealed that compared with dendritic cells, these M2 macrophages displayed a tolerogenic phenotype reflected by a lower expression of costimulatory molecules, an altered balance in IL-12p70 and IL-10 production, and a poor capacity to stimulate T cell proliferation and IFN-γ production. Notably, upon cognate interaction with Th1 cells, these tumor-induced M2 macrophages could be switched to activated M1-like macrophages that expressed high levels of costimulatory molecules, produced high amounts of IL-12 and low amounts of IL-10, and acquired the lymphoid homing marker CCR7. The effects of the interaction between M2 macrophages and Th1 cells could partially be mimicked by activation of these APCs via CD40 in the presence of IFN-γ. Our data on the presence, induction, and plasticity of tumor-induced tolerogenic APCs in cervical cancer suggest that tumor-infiltrated Th1 cells can stimulate a tumor-rejecting environment by switching M2 macrophages to classical proinflammatory M1 macrophages.

Abstract 4600: Green tea polyphenols causes cell cycle arrest and apoptosis of prostate cancer cells by suppressing class I histone deacetylases

Abstract Green Tea polyphenols (GTP) possess anticancer activity and causes cell cycle arrest and apoptosis in a majority of cancer cells however the mechanism(s) involved in this regulation is still unclear. Recent studies from our group [Int J Cancer 126:2520-33, 2010] reported that GTP has ability to alter gene expression by epigenetic modifications including acetylation/deacetylation of histones. Histone acetyltranferases (HATs) and deacetylases (HDACs) enzymes play important role in these modifications. Acetylation relaxes chromatin, making it accessible to transcription factors and increased gene expression. Deacetylation, on the contrary, represses gene expression. HDACs are grouped into 4 classes: class I (HDACs1-3 &amp; 8), class II (HDACs 4-7 and HDACs 9-10), class III (Sirt1-Sirt7) and class IV (HDAC11). Class I HDACs are overexpressed in many cancer types including prostate cancer and their inhibition leads to cell cycle arrest and apoptosis. Since many dietary agents have been reported to cause cell cycle arrest and apoptosis in cancer cells by inhibiting class I HDACs, we propose similar mechanism of action by GTP. Androgen-responsive human prostate cancer LNCaP (harboring WT p53) and androgen-refractory PC-3 (lacking p53) cells were treated with different doses of GTP (10-80µg/ml) for 24h and expression of HDAC1, 2, 3 and 8, p53, p21, Bax and Bcl2 at protein and message levels were determined. Treatment with GTP resulted in dose-dependent decrease in the expression of HDAC 2, 3 and 8 in both prostate cancer cells, but not in HDAC 1, however total HDAC activity was decreased at the highest dose of 80µg/ml GTP. Subsequently, increased acetylation of histone H3 at Lys 9/18 and Histone H4 was observed after GTP treatment. GTP treatment resulted in increased expression of p21and Bax and decrease in Bcl2 at protein and message level in these cells. These alterations were consistent with the G1 phase arrest in dose- and time-dependent manner (69%-80% in LNCaP cells and 40%-62% in PC-3 cells) after GTP treatment. Exposure of cells with GTP up to 72 h resulted in the induction of apoptosis in both cell lines (2.7-84.5% in LNCaP cells and 4.0-85.2% in PC3 cells) as measured by annexin V assay. Furthermore, treatment of cells with proteasomal inhibitor, MG132 together with GTP prevented degradation of class I HDACs, compared to cells treated with GTP alone, indicating increased proteasomal degradation of class I HDACs by GTP. Taken together, we conclude that inhibition of class I HDACs and increased acetylation of histone proteins by GTP contributes to the mechanism of GTP-induced cell cycle arrest and apoptosis of prostate cancer cells, irrespective of their p53 status. 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 4600. doi:10.1158/1538-7445.AM2011-4600

Abstract 2063: Fate of hypoxic cancer cells in microscopic and fully developed macroscopic tumors

Abstract We previously reported, in mouse cancer models of human colorectal cancer HT29 cells, microscopic tumors (&amp;lt;1 mm) were severe hypoxia and poor perfusion, in contrast, larger ones (1-4 mm) were well-perfused and not significantly hypoxic, as tumors increased in size beyond this level, the typical pattern of macroscopic tumor hypoxia reappeared (Cancer Res, 2007; 67: 7646). The fate of hypoxic cells at each stage of cancer development is largely unknown. This issue is critically important for planning anticancer therapy; hypoxia induces cancer therapy resistance. Methods: We injected HT29 cells into peritoneal cavity, ascites tumors (floating in ascites, in dormant status and &amp;lt;1mm in diameter) were evident 4-5 wk later, ascites tumors were harvested 1-7 d followed exogenous hypoxia marker pimonidazole (PIMO) administration, the spatial localization of PIMO adducts visualized by histochemical staining and their relating to viable cells or cell debris in necrosis was used to estimate the lifespan of hypoxic cancer cells, fate of hypoxic cells was also observed in fully developed HT29 subcutaneous xenografts (∼10 mm in diameter). Changes in hypoxia during early stage of tumor development were investigated in an intradermal tumor model using dual hypoxia markers staining technique: various days after cancer cells inoculation (i.e. various size of tumors), we injected PIMO to hosts 1h before their sacrifice to label “current” hypoxic cells, this compared with hypoxia induced protein carbonic anhydrase 9 (CA9) expression, CA9 is able to mark “historic” hypoxia because it has 3 days half-life even after reoxygenation. Stroma formation and angiogenesis were also observed. Results: PIMO adducts closely associated with viable cancer cells in dormant ascites tumors 7 d followed PIMO injection, there was little necrotic tissue in ascites tumors. However, Major PIMO adducts were in necrotic zones 2-3 d later in fully developed macroscopic xenografts. Changes in hypoxia during early stage of tumor development were investigated: 3 to 5 d after tumor initiation, a sole cluster of pure cancer cells (&amp;lt;1 mm) was found, majority of cancer cells had high PIMO binding and high CA9 expression. Seven days after cell inoculation when tumors reached to 2-3 mm in diameter, cancer cells had little PIMO binding but still strong CA9 expression, abundant stroma along with functional blood vessels were evident in the tumors, Which indicated previous hypoxic cells had been recently oxygenated due to stroma formation/angiogenesis switch. Conclusion: Hypoxic cancer cells in submillimeter dormant microscopic tumors have very long lifespan, which maybe oxygenate after angiogenesis and tumor growth. Hypoxic cell in fully developed tumors generally go to necrosis. Hypoxia is critical important in microscopic tumors, hypoxic cells may resist cancer therapies and microscopic tumors develop to macroscopic tumors in a future time. 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 2063. doi:10.1158/1538-7445.AM2011-2063