Tumor Cell Immortalization Research Articles

Cellular Therapies for the Treatment of Hematological Malignancies; Swine Are an Ideal Preclinical Model

The absence of clinically relevant large animal tumor models has historically forced experimental cellular therapies for hematological malignancies to translate directly from murine models to clinical trials. However, recent advances highlight swine as an ideal large animal model to demonstrate the safety of murine proof of concept studies prior to their implementation clinically. The availability of the MHC defined MGH miniature swine herd has been key for the development of novel approaches for hematopoietic cell and solid organ transplantation. New spontaneously arising hematological malignancies in these swine, specifically myeloid leukemias and B cell lymphomas, resemble human malignancies, which has allowed for development of immortalized tumor cell lines and has implications for the development of a large animal transplantable tumor model. The novel development of a SCID swine model has further advanced the field of large animal cancer models, allowing for engraftment of human tumor cells in a large animal model. Here, we will highlight the advantages of the swine pre-clinical model for the study of hematological malignancies. Further, we will discuss our experience utilizing spontaneously arising tumors in MGH swine to create a transplantable tumor model, describe the potential of the immunodeficient swine model, and highlight several novel cellular and biological therapies for the treatment of hematological malignancies in swine as a large animal pre-clinical bridge.

Telomere elongation via alternative lengthening of telomeres (ALT) and telomerase activation in primary metastatic medulloblastoma of childhood.

Elongation of telomeres is necessary for tumor cell immortalization and senescence escape; neoplastic cells use to alternative pathways to elongate telomeres: telomerase reactivation or a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT). Telomerase and ALT pathway has been explored in adult and pediatric gliomas and medulloblastomas (MDBs); however, these mechanisms were not previously investigated in MDBs metastatic at the onset. Therefore, we analyzed the activation of telomerase and ALT pathway in a homogenous cohort of 43 pediatric metastatic medulloblastomas, to investigate whether telomere elongation could play a role in the biology of metastatic MDB. We evaluated telomeres length via telomere-specific fluorescence in situ hybridization (Telo-FISH); we assessed nuclear expression of ATRX by immunohistochemistry (IHC). H3F3A and TERT promoter mutations were analyzed by pyrosequencing, while UTSS methylation status was analyzed via methylation-specific-PCR (MS-PCR). H3F3A mutations were absent in all MDBs, 30% of samples showed ATRX nuclear loss, 18.2% of cases were characterized by TERT promoter mutations, while 60.9% harboured TERT promoter hyper-methylation in the UTSS region. Elongation of telomeres was found in 42.8% of cases. Metastatic MDBs control telomere elongation via telomerase activation (10.7%), induced by TERT promoter mutations in association with UTSS hyper-methylation, and ALT mechanism (32.1%), triggered by ATRX inactivation. Among non-metastatic MDBs, only 5.9% (1/17) showed ATRX nuclear loss with activation of ALT. Our metastatic cases frequently activate ALT pathway, suggesting that it is a common process for senescence escape in primary metastatic medulloblastomas. Furthermore, the activation of mechanisms for telomere elongation is not restricted to certain molecular subgroups in this high-risk group of MDBs.

Contributory role of microRNAs in anti-cancer effects of small molecule inhibitor of telomerase (BIBR1532) on acute promyelocytic leukemia cell line

Telomerase-mediated immortalization and proliferation of tumor cells is a promising anti-cancer treatment strategy and development of potent telomerase inhibitors is believed to open new window of treatments in human malignancies. In the present study, we found that BIBR1532, a small molecule inhibitor of human telomerase, exerted cytotoxic effects on a panel of human cancer cells spanning from solid tumors to hematologic malignancies; however, as compared with solid tumors, leukemic cells were more sensitive to this inhibitor. This was independent of molecular status of p53 in the leukemic cells. The results of a miRNA PCR array revealed that BIBR1532-induced cytotoxic effects in NB4, the most sensitive cell line, was coupled with alteration in a substantial number of cancer-related miRNAs. Interestingly, most of these miRNAs were found to act as tumor suppressors with validated targets in cell cycle or nuclear factor (NF)-κB–mediated apoptosis. In accordance with a bioinformatics analysis, our experimental studies showed that BIBR1532-induced apoptosis is mediated, at least partly, by inhibition of NF-κB. Moreover, we found that the alteration in the expression of miRNAs was coupled with the alteration in the cell cycle progression. To sum up with, a straightforward interpretation of our results is that telomerase inhibition using BIBR1532 not only induced CDKN1A-mediated G1 arrest in NB4, but also resulted in a caspase-3-dependent apoptotic cell death mostly through suppression of NF-κB axis.

Regulatory T Cells and Their Clinical Applications in Antitumor Immunotherapy

Abstract Cancer is a potentially life-threatening disease characterized by the immortalization of tumor cells in the host. Immunotherapy has recently gained increasing interest among researchers due to its tremendous potential for preventing tumor progression and metastasis. Regulatory T cells (Tregs) are a subgroup of suppressive CD4+ T cells that play a vital role in the maintenance of host immune homeostasis. Treg deficiency can induce severe autoimmune, hypersensitivity, and auto-inflammatory disorders, among other diseases. Tregs are commonly enriched in a tumor microenvironment, and a greater number of immune-suppressive Tregs often indicates a poorer prognosis; therefore, there is renewed interest in the function of Tregs and in their clinical application in antitumor immunotherapy. Accumulating strategies that focus on the depletion of Tregs have appeared to be effective in antitumor immunity. It is expected that Treg-targeting strategies will provide great opportunities for improving antitumor efficiency in combination with other therapeutics (e.g., chimeric antigen receptor T cell (CAR-T)-based cell therapy or immune checkpoint blockading).

Abstract 3467: A DAXX-KIFC3 fusion potentiates alternative lengthening of telomeres in osteosarcoma

Abstract Proliferating cells must enact a program of telomere lengthening to counteract the chromosome end replication problem. In most types of cancer cells, telomeres are maintained through the action of the ribonucleoprotein telomerase, but some cancer cells, particularly those of mesenchymal origin, utilize an alternative method of telomere repair and lengthening termed the alternative lengthening of telomeres (ALT) pathway. Since telomere maintenance is essential for tumor cell immortality, better understanding of the ALT mechanism could potentially reveal drug targets that could be used to develop novel therapies for tumors that use ALT. It has been previously observed that ALT tumors frequently carry mutations in ATRX, which partners with the protein DAXX in a chromatin remodeling complex, but how these mutations facilitate the ALT pathway is not well understood. Work in our lab identified an ALT-positive osteosarcoma cell line, identified here as OS1, in which DAXX has undergone a fusion event with the non-canonical kinesin KIFC3. We find that knockdown of the DAXX-KIFC3 fusion neither impairs ALT nor cell proliferation, suggesting that the fusion represents a loss of function. Furthermore, inducible restoration of wild-type DAXX, reversibly abrogates ALT function in this cell line. One of the hallmarks of ALT is localization of telomeres and DNA recombination machinery to nuclear PML bodies, resulting in formation of ALT- associated PML Bodies, or APBs. Thus it may be considered that changes in PML body composition represent a key aspect of the ALT mechanism. We observe that in OS1 both DAXX and ATRX fail to localize to PML bodies. This finding is consistent with the fact that the DAXX-KIFC3 fusion results in loss of a C-terminal SUMO interaction motif that normally mediates PML body interaction. Leveraging our inducible system, using biochemical and imaging approaches, we are working to define the role of DAXX in maintaining PML body composition. Citation Format: Sarah F. Clatterbuck Soper, Soyeon A. Showman, Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sven Bilke, Paul S. Meltzer. A DAXX-KIFC3 fusion potentiates alternative lengthening of telomeres in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2017-3467

Isolation and Characterization of a Distinct Subpopulation from the WM115 Cell Line That Resembles In Vitro Properties of Melanoma Cancer Stem Cells

Despite key advances in cancer therapies, malignant tumors, such as melanoma, continue to be one of the leading causes of mortality. Recent debate on whether cancer can originate from a tumor-initiating subpopulation has permeated oncology and stem cell research. It has been well established that primary and immortalized tumor cells consist of heterogeneous cell populations. The profound effect of tumor heterogeneity on tumor growth and drug resistance remains elusive, but it is highly likely that subpopulations of cancer cells have different capabilities of self-renewal and drug resistance. Discrepancies between excellent in vitro potency and efficacy and poor patient response have been observed on multiple cancer therapeutics. Although this observation can be attributed to many factors, a better understanding of the contribution from subpopulations within a cancer will help bridge the gap between in vitro assay results and patient prognosis. To comprehend this impact, it is critical to isolate and characterize cancer subpopulations that possess higher growth and drug resistance properties so that novel therapeutics can be developed to eventually eradicate all cancer cells. In this article, we describe a method to enrich a subpopulation, CB4, from the melanoma cell line WM115. CB4 exhibited higher anchorage-independent growth, higher survival under serum starvation condition, and lower drug sensitivity to commonly used melanoma treatment compared with WM115. Details of functional properties and gene expression of CB4 compared with WM115 are reported. Our study demonstrates that it is feasible to isolate and enrich a subpopulation that exhibits higher growth capacity and treatment resistance from an immortalized tumor cell line.

Glycogen synthase kinase-3β is a pivotal mediator of cancer invasion and resistance to therapy.

Tumor cell invasion and resistance to therapy are the most intractable biological characteristics of cancer and, therefore, the most challenging for current cancer research and treatment paradigms. Refractory cancers, including pancreatic cancer and glioblastoma, show an inextricable association between the highly invasive behavior of tumor cells and their resistance to chemotherapy, radiotherapy and targeted therapies. These aggressive properties of cancer share distinct cellular pathways that are connected to each other by several molecular hubs. There is increasing evidence to show that glycogen synthase kinase (GSK)‐3β is aberrantly activated in various cancer types and this has emerged as a potential therapeutic target. In many but not all cancer types, aberrant GSK3β sustains the survival, immortalization, proliferation and invasion of tumor cells, while also rendering them insensitive or resistant to chemotherapeutic agents and radiation. Here we review studies that describe associations between therapeutic stimuli/resistance and the induction of pro‐invasive phenotypes in various cancer types. Such cancers are largely responsive to treatment that targets GSK3β. This review focuses on the role of GSK3β as a molecular hub that connects pathways responsible for tumor invasion and resistance to therapy, thus highlighting its potential as a major cancer therapeutic target. We also discuss the putative involvement of GSK3β in determining tumor cell stemness that underpins both tumor invasion and therapy resistance, leading to intractable and refractory cancer with dismal patient outcomes.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.

CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.

Telomere recombination and the ALT pathway: a therapeutic perspective for cancer.

Telomeres are essential for cell proliferation and tumor cell immortalization requires the presence of a telomere maintenance mechanism. Thus, interfering with this mechanism constitutes a potential means to impede cell proliferation and tumor progression. Many cancer cells rely on telomerase activity to ensure indefinite proliferation capacity and developing therapeutic approaches that target telomerase has attracted much attention in the last couple of decades. Nevertheless, a non-negligible proportion of tumors utilize telomerase- independent, alternative mechanisms to lengthen telomeres (ALT). Here we briefly discuss both our current understanding of ALT mechanisms and the potential to develop a therapeutic approach targeting ALT.

Genome editing in human stem cells.

The use of custom-engineered sequence-specific nucleases (including CRISPR/Cas9, ZFN, and TALEN) allows genetic changes in human cells to be easily made with much greater efficiency and precision than before. Engineered double-stranded DNA breaks can efficiently disrupt genes, or, with the right donor vector, engineer point mutations and gene insertions. However, a number of design considerations should be taken into account to ensure maximum gene targeting efficiency and specificity. This is especially true when engineering human embryonic stem or induced pluripotent stem cells (iPSCs), which are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe a protocol for easily engineering genetic changes in human iPSCs, through which we typically achieve targeting efficiencies between 1% and 10% without any subsequent selection steps. Since this protocol only uses the simple transient transfection of plasmids and/or single-stranded oligonucleotides, most labs will easily be able to perform it. We also describe strategies for identifying, cloning, and genotyping successfully edited cells, and how to design the optimal sgRNA target sites and donor vectors. Finally, we discuss alternative methods for gene editing including viral delivery vectors, Cas9 nickases, and orthogonal Cas9 systems.

Distribution of TERT promoter mutations in pediatric and adult tumors of the nervous system

Hot spot mutations in the promoter region of telomerase reverse transcriptase (TERT) have recently been described in several human tumor entities. These mutations result in an upregulation of the telomerase complex activity and thus constitute a relevant mechanism for immortalization of tumor cells. Knowledge of the TERT promoter status in tumors is likely to be of interest for molecular classification and as a potential target for therapy. We, therefore, performed a systematic analysis of TERT promoter mutations in 1,515 tumors of the human nervous system and its coverings including 373 pediatric and 1,142 adult patients. We detected a total of 327 mutations. TERT promoter mutations were exceedingly rare in tumors typically encountered in pediatric patients. In entities typically encountered in adult patients TERT promoter mutations were strongly associated with older age (p < 0.0001). Highest mutation frequencies were detected in gliosarcomas (81 %), oligodendrogliomas (78 %), oligoastrocytomas (58 %), primary glioblastomas (54 %), and solitary fibrous tumors (50 %). Related to other molecular alterations, TERT promoter mutations were strongly associated with 1p/19q loss (p < 0.0001), but inversely associated with loss of ATRX expression (p < 0.0001) and IDH1/IDH2 mutations (p < 0.0001). TERT promoter mutations are typically found in adult patients and occur in a highly tumor type-associated distribution.

Abstract 3095: The molecular, cellular, and physiological consequences of disrupting the HIF-1α interaction

Abstract Many cancers contain regions of hypoxia due to rapid cell proliferation and the presence of blood vessels within the tumor that are structurally and functionally abnormal, resulting in the heterogeneity of blood flow. This state of hypoxia is associated with increased risk of treatment failure, metastasis and patient mortality. A principal mechanism by which cancer cells adapt to the hypoxic microenvironment is through the activity of hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor composed of an O2-regulated HIF-1α subunit that dimerizes with a constitutively expressed HIF-1β subunit. HIF-1α expression under hypoxic conditions regulates hundreds of genes including those that play key roles in invasion/metastasis, angiogenesis, tumor cell immortalization, and resistance to chemotherapy and radiation. Therefore, the inhibition of transcription driven by HIF has the potential for cancer treatment. One way to reduce the activity of this protein is by disrupting the complex that HIF forms with p300, an essential transcriptional coactivator. Previous data from our laboratory showed that several members of the epidithiodiketopiperazine (ETP) family of natural products are able to block the interaction between HIF-1α and p300. Here, we extend these studies by examining the comprehensive effect that select ETPs have on the disruption of the HIF/p300 complex at the molecular, cellular, and physiological levels. We began our studies by performing a series of rat aortic ring assays to determine the antiangiogenic effect of selected ETPs, namely chetomin, chaetocin, and gliotoxin. Chetomin and chaetocin concentrations of 50 nM inhibited approximately 90% of outgrowth, while 500 nM of gliotoxin was needed to achieve a similar effect; these compounds had GI50 of 22, 11, and 175 nM, respectively. In ongoing work, we hope to determine if the antiangiogenic effects that were observed in our rat aortic ring assays, are due to disruption of the HIF/p300 complex. To this end we are immunoprecipitating the complex from cells in the absence or presence of the aforementioned ETPs. The molecular consequences of blocking the HIF/p300 interaction will be assayed by qRT-PCR for hypoxia-regulated genes such as VEGF and GLUT1; cell proliferation and migration assays will also be performed to monitor cellular effects. Together, these results will be key in determining the feasibility of targeting this interaction for cancer therapeutics. 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 3095. doi:10.1158/1538-7445.AM2011-3095

Establishment and genetic characterization of an immortal tumor cell line derived from intestinal-type sinonasal adenocarcinoma

Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumor etiologically related to professional exposure to wood dust. The overall prognosis is poor, mainly due to the difficulty to resect the tumor completely in this anatomically complex region. Therefore, there is great need for alternative treatments. However, the lack of a good tumor model system for ITAC has hampered the development and testing of new therapeutic agents. Here, we report the establishment and characterization of the first human ITAC cell line named ITAC-3. The cell line was initiated from small explants of a T4bN0M0 colonic type ITAC from the ethmoid sinus. Growth and invasion parameters as well as genetic characteristics were analyzed. The population doubling time was 18 h and the cell line was capable of invasion in matrigel. Chromosomal analysis showed a tetraploid karyotype with both numerical and structural aberrations. High resolution microarray CGH analysis identified many copy number alterations, including homozygous deletions. TP53 carried a mutation c.818G>T in exon eight concurring with a strong nuclear protein overexpression. Immunohistochemical analysis showed protein overexpression of EGFR and normal expression of β-catenin and p16. This is the first report of the establishment of a cell line derived from a primary ITAC. The genomic profile of the cell line was the same as the primary tumor from which it was derived. This new cell line will be a useful tool for the development and testing of new therapeutic agents for this tumor type.

Parimal Chowdhury’s work on smoking related pancreatic disorders

Cigarette smoking is a known risk factor for the development of numerous diseases. The role of nicotine in the induction of pancreatic inflammation and pancreatic cancer as a result of cigarette smoking has been recognized and reported in the literature. The mechanism by which nicotine induces such pathologies is as yet unknown. An understanding of the proliferative potential of nicotine in primary and tumor cells of the pancreas will allow us to develop measures that will ultimately lead to intervention, prevention and treatment of these diseases. Studies show that nicotine can increase the cell numbers of cer-tain cancer cell lines, suggesting that exposure to nicotine can lead to the disruption of the dynamic balance between cell death and proliferation, which is required for normal functioning of cells. We hypothesize that nicotine induces oxidative stress in pancreatic acinar cells and thus contributes to this disruption. We have used the AR42J cell line in our study because of its stability as an immortal tumor cell line and its known physiological similarity to primary acinar cells. Our studies show that mitogen activated protein kinase signaling is induced by nicotine in AR42J cells, causing an increase in lipid peroxidation and a subsequent decrease in cell function. Our data suggest that exposure to nicotine induces oxidative stress, leading to cell injury and compromised function, thus implicating cigarette smoking as a plausible mechanism.

Therapy-induced selective loss of leukemia-initiating activity in murine adult T cell leukemia

Chronic HTLV-I (human T cell lymphotropic virus type I) infection may cause adult T cell leukemia/lymphoma (ATL), a disease with dismal long-term prognosis. The HTLV-I transactivator, Tax, initiates ATL in transgenic mice. In this study, we demonstrate that an As(2)O(3) and IFN-α combination, known to trigger Tax proteolysis, cures Tax-driven ATL in mice. Unexpectedly, this combination therapy abrogated initial leukemia engraftment into secondary recipients, whereas the primary tumor bulk still grew in the primary hosts, only to ultimately abate later on. This loss of initial transplantability required proteasome function. A similar regimen recently yielded unprecedented disease control in human ATL. Our demonstration that this drug combination targeting Tax stability abrogates tumor cell immortality but not short-term growth may foretell a favorable long-term efficiency of this regimen in patients.

MOS, aneuploidy and the ploidy cycle of cancer cells

After DNA or spindle damage, p53-defective tumor cells undergo a complex cycle of reversible polyploidy. How this process occurs and more importantly, why, has recently become the focus of several research groups, prompting this review in which we discuss two related phenomena that accompany the reversible polyploidy of tumor cells: the induction of meiosis genes such as MOS and the decrease in genomic instability observed during the reversion from polyploidy to para-diploidy. The reversible polyploidy likely provides the means through which the balance between increased chromosome instability (CIN), driving genetic variation and decreased CIN, necessary for perpetuating these malignant clones, is maintained. These concepts are integrated with recent findings that many meiotic and self-renewal genes become activated during reversible polyploidy and lead us to the hypothesis that tumor cell immortality may be achieved through germline-like transmission.

Abstract 3215: Expression of hTERT predicts immortalization of glioma-derived primary cell cultures and is associated with shorter overall survival in glioblastoma patients

Abstract hTERT represents the catalytic subunit of telomerase leading to cell immortalization by stabilizing telomere lengths. Aim of this study was to characterise hTERT expression in gliomas and the derived cell cultures with a focus on glioblastomas (GBM) and to investigate its relation with tumor cell immortalization in vitro and disease progression in vivo. During the last nine years primary cell cultures were established from 265 tumor tissues obtained during surgery and histologically verified according to WHO criteria as low grade glioma WHO I/II (n=22) and anaplastic astrocytoma WHO III (n=15), GBM WHO IV (n=219), gliosarcoma (n=7) and giant cell glioblastoma (n=2). hTERT gene expression was investigated in all primary cell cultures and additionally in GBM tumor tissues (n=80) by RT-PCR. hTERT mRNA expression levels were calculated relatively to GAPDH mRNA amplified concomitantly and in subgroups verified by real-time RT-PCR. Telomerase enzyme activity was confirmed using the Telomeric Repeat Amplification Protocol (TRAP) of the TRAPeze Telomerase Detection Kit (Chemicon). hTERT expression data were compared to overall survival of GBM patients. Thirty-one percent (81/265) of the analysed cell cultures displayed hTERT gene expression. All of the hTERT-expressing primocultures, which were exclusively high grade gliomas (WHO III/ IV), developed into stable, immortal cell lines whereas hTERT negative cells failed to grow in vitro or ceased growth between passages 1 to 10. In contrast, all low grade gliomas (WHO I/II; n=22) were negative with respect to hTERT expression and characterised by limited in vitro cultivation demonstrating the less aggressive potential of these tumors. In parallel in vivo hTERT expression was analysed in 80 GBM tumor samples. 37/80 (46%) showed detectable hTERT expression and 54% (43/80) were negative with respect to hTERT. Kaplan-Meier survival estimates revealed a significant survival benefit for patients whose tumors lacked hTERT expression (p&amp;lt; 0.04) with a median survival of 20.1 months versus 14.3 months for patients whose tumors expressed hTERT. Summing up our data show that a) telomerase activity, reflected by hTERT expression is essential for successful in vitro growth of brain glioma-derived primocultures/cell lines. Thus in vitro studies on GBM generally focus exclusively on the hTERT-positive patient subgroup. b) hTERT gene expression can predict the potential to establish a stable, immortal cell model from a brain tumor specimen. Focussing on hTERT expression in vivo our data revealed that hTERT expression is associated with significantly shorter overall survival in GBM patients. Thus hTERT might have quality as prognostic biomarker predicting tumor aggressiveness. 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 3215.

Abundance of a distinct cluster of telomere t-stumps in advanced breast cancer cell line

Breast tumors are the second major cause of cancer-related death in women worldwide. These tumors are aggressive, leading to metastatic cancers that are heterogeneous in nature, with numerous subtypes. The basal-like tumor subtype invariably shows unfavorable prognosis and is often characterized by the lack of estrogen, progesterone and HER2 receptors. These cancer types do not respond to the current targeted therapies. Therefore, the need for the discovery of novel diagnostic markers/therapeutic targets is of paramount importance. Immortalization of breast tumor cells leading to advanced stage cancer is one of the pivotal steps in breast cancer and telomeres/telomerase play a critical role in this process. Using single telomere length analysis, cell lines with a basal-like phenotype encompassing immortalized/non-tumorigenic MCF10A and invasive/metastatic MCF10CA1 along with the MCF-7 cell line were examined for the presence of a unique class of telomere t-stumps. Telomerase activity, protein levels of telomerase and bulk telomere lengths were assessed in the above-mentioned cell lines. This is the first study describing the existence of a distinct class of extremely short telomeres termed 't-stumps' in breast cancer cell lines. The cell lines MCF10A and MCF10CA1 showed distinct telomeric bands in the molecular size range of 100-1,000 bp, whereas the MCF-7 cell line showed very low levels of t-stumps. Of note is that only the highly invasive/metastatic cancer cell line MCF10CA1 exhibited an abundance of a cluster of t-stumps with a size distribution range of 500-700 bp. These unique t-stumps observed in the advanced breast cancer cell line may serve as a novel diagnostic marker and also form a key molecular target for novel anticancer therapy.

Molecular Characterization of Putative Chordoma Cell Lines

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.