bluuebunny/crossref_metadata_2025 huggingface dataset:
Premature senescence is a major response to DNA cross-linking agents in BRCA1-defective cells: implication for tailored treatments of BRCA1 mutation carriers
Authors: Santarosa Manuela , Del Col Laura , Tonin Elena , Caragnano Angela , Viel Alessandra , Maestro Roberta
Published: April 2009

BRCA1-associated tumors are characterized by an elevated genomic instability and peculiar expression profiles. Nevertheless, tailored treatments for BRCA1 mutation carriers have only been partially investigated up to now. The implementation of therapeutic strategies specific for these patients has been in part hindered by the paucity of proper preneoplastic and neoplastic BRCA1-deficient tumor cell models. In this study, we took advantage of the RNA interference technology to generate a series of partially transformed (HBL100) and tumorigenic (MCF7 and T47D) breast cancer cell lines in which BRCA1 expression was silenced at different levels. These cell models were probed by clonogenic assay for their response to several DNA-damaging agents commonly used in cancer therapy (mitomycin C, cisplatin, doxorubicin, and etoposide). Our models confirmed the peculiar sensitivity to interstrand cross-link inducers associated with BRCA1 deficiency. Intriguingly, the increased sensitivity to these compounds displayed by BRCA1-defective cells was not correlated with the extent of apoptotic cell death but rather associated to an increased fraction of growth-arrested, enlarged, multinucleated β-galactosidase–positive senescent cells. Overall, our results support a role for BRCA1 in the regulation of interstrand cross-link–induced premature senescence and suggest a reconsideration of the therapeutic power of mitomycin/platinum-based treatments in BRCA1 carriers. Moreover, our data further prompt the setup of strategies for the imaging of the senescence response in vivo. [Mol Cancer Ther 2009;8(4):844–54]
KRAS/BRAF mutation status and ERK1/2 activation as biomarkers for MEK1/2 inhibitor therapy in colorectal cancer
Authors: Yeh Jen Jen , Routh Elizabeth D. , Rubinas Tara , Peacock Janie , Martin Timothy D. , Shen Xiang Jun , Sandler Robert S. , Kim Hong Jin , Keku Temitope O. , Der Channing J.
Published: April 2009

Phase II clinical trials of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors are ongoing and ERK1/2 activation is frequently used as a biomarker. In light of the mutational activation of BRAF and KRAS in colorectal cancer, inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase are anticipated to be promising. Previous studies in pancreatic cancer have found little correlation between BRAF/KRAS mutation status and ERK1/2 activation, suggesting that identifying biomarkers of MEK inhibitor response may be more challenging than previously thought. The purpose of this study was to evaluate the effectiveness of MEK inhibitor therapy for colorectal cancer and BRAF/KRAS mutation status and ERK1/2 activation as biomarkers for MEK inhibitor therapy. First, we found that MEK inhibitor treatment impaired the anchorage-independent growth of nearly all KRAS/BRAF mutant, but not wild-type, colorectal cancer cells. There was a correlation between BRAF, but not KRAS, mutation status and ERK1/2 activation. Second, neither elevated ERK1/2 activation nor reduction of ERK1/2 activity correlated with MEK inhibition of anchorage-independent growth. Finally, we validated our cell line observations and found that ERK1/2 activation correlated with BRAF, but not KRAS, mutation status in 190 patient colorectal cancer tissues. Surprisingly, we also found that ERK activation was elevated in normal colonic epithelium, suggesting that normal cell toxicity may be a complication for colorectal cancer treatment. Our results suggest that although MEK inhibitors show promise in colorectal cancer, KRAS/BRAF mutation status, but not ERK activation as previously thought, may be useful biomarkers for MEK inhibitor sensitivity. [Mol Cancer Ther 2009;8(4):834–43]
The selective hypoxia inducible factor-1 inhibitor PX-478 providesin vivoradiosensitization through tumor stromal effects
Authors: Schwartz David L. , Powis Garth , Thitai-Kumar Arun , He Yi , Bankson James , Williams Ryan , Lemos Robert , Oh Junghwan , Volgin Andrei , Soghomonyan Suren , Nishii Ryuichi , Alauddin Mian , Mukhopadhay Uday , Peng Zhenghong , Bornmann William , Gelovani Juri
Published: April 2009

Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element–specific micro–positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1–dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms. [Mol Cancer Ther 2009;8(4):947–58]
HDAC2 regulates chromatin plasticity and enhances DNA vulnerability
Authors: Marchion Douglas C. , Bicaku Elona , Turner Joel G. , Schmitt Morgen L. , Morelli Dawn R. , Munster Pamela N.
Published: April 2009

Histone deacetylases (HDAC) may have a prominent role in the development of cancer and the response to anticancer therapy. However, the therapeutic relevance and tissue specificity of individual HDAC enzymes remain largely unknown. HDAC inhibitors may function as sensitizing agents to chemotherapies that target DNA through their effects on chromatin structure and plasticity. Here, we report a new role for HDAC2 as a regulator of chromatin compaction status and the mediator of HDAC inhibitor-induced sensitization to chemotherapy. The selective depletion of HDAC2 by small interfering RNA led to reduced expression of heterochromatin maintenance proteins and morphologic changes indicative of chromatin decondensation. Furthermore, depletion of HDAC2 but not HDAC1 or HDAC6 was sufficient to sensitize breast cancer cells to topoisomerase inhibitor-induced apoptosis. The levels of HDAC2 expression appear to correlate with the degree of HDAC inhibitor-induced histone acetylation in a surrogate tissue in patients. These data suggest that HDAC2 may be a relevant pharmacologic and biological target for combination therapy involving drugs that target DNA.[Mol Cancer Ther 2009;8(4):794–801]
Targeting the sphingolipid metabolism to defeat pancreatic cancer cell resistance to the chemotherapeutic gemcitabine drug
Authors: Guillermet-Guibert Julie , Davenne Lise , Pchejetski Dimitri , Saint-Laurent Nathalie , Brizuela Leyre , Guilbeau-Frugier Céline , Delisle Marie-Bernadette , Cuvillier Olivier , Susini Christiane , Bousquet Corinne
Published: April 2009

Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.[Mol Cancer Ther 2009;8(4):809–20]
Alterations in the Noxa/Mcl-1 axis determine sensitivity of small cell lung cancer to the BH3 mimetic ABT-737
Authors: Hauck Paula , Chao Bo H. , Litz Julie , Krystal Geoffrey W.
Published: April 2009

To understand the molecular basis for variable sensitivity to the BH3 mimetic drug ABT-737, the abundance of Bcl-2 family members was assayed in a panel of small cell lung cancer cell lines whose sensitivity varied over a 2-log range. Elevated Noxa and Bcl-2 levels directly correlated with sensitivity to ABT-737, whereas Mcl-1 levels were similar in all cell lines tested regardless of sensitivity. Transgenically enforced expression of Noxa but not Bcl-2 resulted in increased sensitivity to ABT-737 in multiple cell lines. This increase was especially pronounced in the H209 cell line in which expression of Noxa resulted in a proportionate decline in Mcl-1 expression. Although overexpression of Noxa enhanced sensitivity of the H526 and H82 cell lines to ABT-737, it did not result in altered Mcl-1 levels. Similarly, small interfering RNA–mediated knockdown of Noxa expression in the H146 cell line, which increased resistance to ABT-737, did not result in altered Mcl-1 levels. Therefore, three of four cell lines studied failed to show Noxa-mediated regulation of Mcl-1 expression. However, despite failure to regulate Mcl-1 levels, Noxa blocked binding of Bim to Mcl-1 following its release from Bcl-2 by ABT-737. Finally, we observed that a 24-hour incubation of the H526 and WBA cell lines with ABT-737 resulted in increased Noxa expression, suggesting that Noxa may play a direct role in ABT-737–mediated apoptosis. These results indicate that Noxa expression is the critical determinant of ABT-737 sensitivity and loss of Noxa-mediated regulation of Mcl-1 expression may be an important feature of small cell lung cancer biology. [Mol Cancer Ther 2009;8(4):883–92]
Ataxia telangiectasia and rad3-related kinase contributes to cell cycle arrest and survival after cisplatin but not oxaliplatin
Authors: Lewis Kriste A. , Lilly Kia K. , Reynolds Evelyn A. , Sullivan William P. , Kaufmann Scott H. , Cliby William A.
Published: April 2009

The DNA cross-linking agents cisplatin and oxaliplatin are widely used in the treatment of human cancer. Lesions produced by these agents are widely known to activate the G1 and G2 cell cycle checkpoints. Less is known about the role of the intra–S-phase checkpoint in the response to these agents. In the present study, two different cell lines expressing a dominant-negative kinase dead (kd) version of the ataxia telangiectasia and rad3-related (ATR) kinase in an inducible fashion were examined for their responses to these two platinating agents and a variety of other DNA cross-linking drugs. The expression of the kdATR allele markedly sensitized the cells to cisplatin, but not to oxaliplatin, as assessed by inhibition of colony formation, induction of apoptosis, and cell cycle analysis. Similar differences in survival were noted for melphalan (ATR dependent) and 4-hydroperoxycyclophosphamide (ATR independent). Further experiments showed that ATR function is not necessary for removal of Pt-DNA adducts. The predominant difference between the responses to the two platinum drugs was the presence of a drug-specific ATR-dependent S-phase arrest after cisplatin but not oxaliplatin. These results indicate that involvement of ATR in the response to DNA cross-linking agents is lesion specific. This observation might need to be taken into account in the development and use of ATR or Chk1 inhibitors. [Mol Cancer Ther 2009;8(4):855–63]
Niacin status and treatment-related leukemogenesis
Authors: Kirkland James B.
Published: April 2009

Chemotherapy often causes damage to hematopoietic tissues, leading to acute bone marrow suppression and the long term development of leukemias. Niacin deficiency, which is common in cancer patients, causes dramatic genomic instability in bone marrow cells in an in vivo rat model. From a mechanistic perspective, niacin deficiency delays excision repair and causes double strand break accumulation, which in turn favors chromosome breaks and translocations. Niacin deficiency also impairs cell cycle arrest and apoptosis in response to DNA damage, which combine to encourage the survival of cells with leukemogenic potential. Conversely, pharmacological supplementation of rats with niacin increases bone marrow poly(ADP-ribose) formation and apoptosis. Improvement of niacin status in rats significantly decreased nitrosourea-induced leukemia incidence. The data from our rat model suggest that niacin supplementation of cancer patients may decrease the severity of short- and long-term side effects of chemotherapy, and could improve tumor cell killing through activation of poly(ADP-ribose)-dependent apoptosis pathways. [Mol Cancer Ther 2009;8(4):725–32]
DNA-Dependent Protein Kinase (DNA-PK)–Dependent Cisplatin-Induced Loss of Nucleolar Facilitator of Chromatin Transcription (FACT) and Regulation of Cisplatin Sensitivity by DNA-PK and FACT
Authors: Dejmek Janna , Iglehart J. Dirk , Lazaro Jean-Bernard
Published: April 2009

Both the Ku subunit of the DNA-dependent protein kinase (DNA-PK) and the facilitator of chromatin transcription (FACT) complex reportedly bind cisplatin-DNA adducts. For this study, we developed an immunocytochemical assay based on detergent extraction allowing unveiling nucleolar subpopulations of proteins present in both the nucleoplasm and the nucleolus. Immunofluorescence analysis in various human cancer cell lines and immunoblotting of isolated nucleoli show that DNA-PK catalytic subunit (DNA-PKcs), Ku86, the Werner syndrome protein (WRN), and the structure-specific recognition protein 1 (SSRP1) subunit of FACT colocalize in the nucleolus and exit the nucleolus after cisplatin treatment. Nucleolar localization of Ku is also lost after γ or UV irradiation and exposure to DNA-damaging drugs, such as actinomycin D, mitomycin C, hydroxyurea, and doxorubicin. Ku86 and WRN leave the nucleolus after exposure to low (>1 μg/mL) doses of cisplatin. In contrast, the SSRP1 association with the nucleolus was disrupted only by high (50-100 μg/mL) doses of cisplatin. Both cisplatin-induced loss of nucleolar SSRP1 and DNA-PK activation are suppressed by pretreatment of the cells with wortmannin or the DNA-PK inhibitor NU7026 but not by the phosphatidylinositol 3-kinase inhibitor LY294002. In the same conditions, kinase inhibitors did not alter the exit of DNA-PKcs and WRN, suggesting that different mechanisms regulate the exit of DNA-PK/WRN and FACT from the nucleolus. Furthermore, RNA silencing of DNA-PKcs blocked the cisplatin-induced exit of nucleolar SSRP1. Finally, silencing of DNA-PKcs or SSRP1 by short hairpin RNA significantly increased the sensitivity of cancer cells to cisplatin.(Mol Cancer Res 2009;7(4):581–91)
Genetic Alterations and Oncogenic Pathways Associated with Breast Cancer Subtypes
Authors: Hu Xiaolan , Stern Howard M. , Ge Lin , O'Brien Carol , Haydu Lauren , Honchell Cynthia D. , Haverty Peter M. , Peters Brock A. , Wu Thomas D. , Amler Lukas C. , Chant John , Stokoe David , Lackner Mark R. , Cavet Guy
Published: April 2009

Breast cancers can be divided into subtypes with important implications for prognosis and treatment. We set out to characterize the genetic alterations observed in different breast cancer subtypes and to identify specific candidate genes and pathways associated with subtype biology. mRNA expression levels of estrogen receptor, progesterone receptor, and HER2 were shown to predict marker status determined by immunohistochemistry and to be effective at assigning samples to subtypes. HER2+ cancers were shown to have the greatest frequency of high-level amplification (independent of the ERBB2 amplicon itself), but triple-negative cancers had the highest overall frequencies of copy gain. Triple-negative cancers also were shown to have more frequent loss of phosphatase and tensin homologue and mutation of RB1, which may contribute to genomic instability. We identified and validated seven regions of copy number alteration associated with different subtypes, and used integrative bioinformatics analysis to identify candidate oncogenes and tumor suppressors, including ERBB2, GRB7, MYST2, PPM1D, CCND1, HDAC2, FOXA1, and RASA1. We tested the candidate oncogene MYST2 and showed that it enhances the anchorage-independent growth of breast cancer cells. The genome-wide and region-specific differences between subtypes suggest the differential activation of oncogenic pathways. (Mol Cancer Res 2009;7(4):511–22)
The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts
Authors: de Vries Teun J. , Mullender Margriet G. , van Duin Marion A. , Semeins Cornelis M. , James Neil , Green Tim P. , Everts Vincent , Klein-Nulend Jenneke
Published: April 2009

Tumor cells in the bone microenvironment are able to initiate a vicious cycle of bone degradation by mobilizing osteoclasts, multinucleated cells specialized in bone degradation. c-Src is highly expressed both in tumors and in osteoclasts. Therefore, drugs like AZD0530, designed to inhibit Src activity, could selectively interfere with both tumor and osteoclast activity. Here we explored the effects of AZD0530 on human osteoclast differentiation and activity. The effect on osteoclasts formed in vivo was assessed in mouse fetal calvarial explants and in isolated rabbit osteoclasts, where it dose-dependently inhibited osteoclast activity. Its effect on formation and activity of human osteoclasts in vitro was determined in cocultures of human osteoblasts and peripheral blood mononuclear cells. AZD0530 was most effective in inhibiting osteoclast-like cell formation when present at the onset of osteoclastogenesis, suggesting that Src activity is important during the initial phase of osteoclast formation. Formation of active phosphorylated c-Src, which was highly present in osteoclast-like cells in cocultures and in peripheral blood mononuclear cell monocultures, was significantly reduced by AZD0530. Furthermore, it reversibly prevented osteoclast precursor migration from the osteoblast layer to the bone surface and subsequent formation of actin rings and resorption pits. These data suggest that Src is pivotal for the formation and activity of human osteoclasts, probably through its effect on the distribution of the actin microfilament system. The reversible effect of AZD0530 on osteoclast formation and activity makes it a promising candidate to temper osteoclastic bone degradation in bone diseases with enhanced osteoclast activity such as osteolytic metastatic bone disease. (Mol Cancer Res 2009;7(4):476–88)
RhoA Regulates G1-S Progression of Gastric Cancer Cells by Modulation of Multiple INK4 Family Tumor Suppressors
Authors: Zhang Siyuan , Tang Qiulin , Xu Feng , Xue Yan , Zhen Zipeng , Deng Yu , Liu Ming , Chen Ji , Liu Surui , Qiu Meng , Liao Zhengyin , Li Zhiping , Luo Deyun , Shi Fang , Zheng Yi , Bi Feng
Published: April 2009

RhoA, a member of the Rho GTPase family, has been extensively studied in the regulation of cytoskeletal dynamics, gene transcription, cell cycle progression, and cell transformation. Overexpression of RhoA is found in many malignancies and elevated RhoA activity is associated with proliferation phenotypes of cancer cells. We reported previously that RhoA was hyperactivated in gastric cancer tissues and suppression of RhoA activity could partially reverse the proliferation phenotype of gastric cancer cells, but the underlying mechanism has yet to be elucidated. It has been reported that RhoA activation is crucial for the cell cycle G1-S procession through the regulation of Cip/Kip family tumor suppressors in benign cell lines. In this study, we found that selective suppression of RhoA or its effectors mammalian Diaphanous 1 and Rho kinase (ROCK) by small interfering RNA and a pharmacologic inhibitor effectively inhibited proliferation and cell cycle G1-S transition in gastric cancer lines. Down-regulation of RhoA-mammalian Diaphanous 1 pathway, but not RhoA-ROCK pathway, caused an increase in the expression of p21Waf1/Cip1 and p27Kip1, which are coupled with reduced expression and activity of CDK2 and a cytoplasmic mislocalization of p27Kip1. Suppression of RhoA-ROCK pathway, on the other hand, resulted in an accumulation of p15INK4b, p16INK4a, p18INK4c, and p19INK4d, leading to reduced expression and activities of CDK4 and CDK6. Thus, RhoA may use two distinct effector pathways in regulating the G1-S progression of gastric cancer cells.(Mol Cancer Res 2009;7(4):570–80)
Mcl-1 Is Required for Melanoma Cell Resistance to Anoikis
Authors: Boisvert-Adamo Karen , Longmate Whitney , Abel Ethan V. , Aplin Andrew E.
Published: April 2009

Melanoma is a particularly aggressive tumor type that exhibits a high level of resistance to apoptosis. The serine/threonine kinase B-RAF is mutated in 50% to 70% of melanomas and protects melanoma cells from anoikis, a form of apoptosis induced by lack of adhesion or adhesion to an inappropriate matrix. Mutant B-RAF down-regulates two BH3-only proapoptotic proteins, BimEL and Bad. BH3-only proteins act, at least in part, by sequestering prosurvival Bcl-2 family proteins and preventing them from inhibiting the mitochondrial apoptotic pathway. Several Bcl-2 proteins are up-regulated in melanoma; however, the mechanisms of up-regulation and their role in melanoma resistance to anoikis remain unclear. Using RNA interference, we show that depletion of Mcl-1 renders mutant B-RAF melanoma cells sensitive to anoikis. By contrast, minor effects were observed following depletion of either Bcl-2 or Bcl-XL. Mcl-1 expression is enhanced in melanoma cell lines compared with melanocytes and up-regulated by the B-RAF-MEK-extracellular signal-regulated kinase 1/2 pathway through control of Mcl-1 protein turnover. Similar to B-RAF knockdown cells, adhesion to fibronectin protected Mcl-1 knockdown cells from apoptosis. Finally, expression of Bad, which does not sequester Mcl-1, further augmented apoptosis in nonadherent Mcl-1 knockdown cells. Together, these data support the notion that BH3 mimetic compounds that target Mcl-1 may be effective for the treatment of melanoma in combinatorial strategies with agents that disrupt fibronectin-integrin signaling. (Mol Caner Res 2009;7(4):549–56)
Physiologic Oxygen Concentration Enhances the Stem-Like Properties of CD133+ Human Glioblastoma Cells In vitro
Authors: McCord Amy M. , Jamal Muhammad , Shankavarum Uma T. , Lang Frederick F. , Camphausen Kevin , Tofilon Philip J.
Published: April 2009

In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell–like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133+ cells sorted from three GB neurosphere cultures at 7% O2 reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1α was not affected in CD133+ TSCs grown at 7% O2, hypoxia-inducible factor 2α was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133+ cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell–like phenotype of CD133+ GB cells. (Mol Cancer Res 2009;7(4):489–97)
Targeting Melanoma with Dual Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitors
Authors: Marone Romina , Erhart Dominik , Mertz Ann C. , Bohnacker Thomas , Schnell Christian , Cmiljanovic Vladimir , Stauffer Frédéric , Garcia-Echeverria Carlos , Giese Bernd , Maira Sauveur-Michel , Wymann Matthias P.
Published: April 2009

Phosphoinositide 3-kinase (PI3K)/protein kinase B/Akt and Ras/mitogen-activated protein kinase pathways are often constitutively activated in melanoma and have thus been considered as promising drug targets. Exposure of melanoma cells to NVP-BAG956, NVP-BBD130, and NVP-BEZ235, a series of novel, potent, and stable dual PI3K/mammalian target of rapamycin (mTOR) inhibitors, resulted in complete G1 growth arrest, reduction of cyclin D1, and increased levels of p27KIP1, but negligible apoptosis. In contrast, treatment of melanoma with the pan-class I PI3K inhibitor ZSTK474 or the mTORC1 inhibitor rapamycin resulted only in minor reduction of cell proliferation. In a syngeneic B16 mouse melanoma tumor model, orally administered NVP-BBD130 and NVP-BEZ235 efficiently attenuated tumor growth at primary and lymph node metastatic sites with no obvious toxicity. Metastatic melanoma in inhibitor-treated mice displayed reduced numbers of proliferating and significantly smaller tumor cells. In addition, neovascularization was blocked and tumoral necrosis increased when compared with vehicle-treated mice. In conclusion, compounds targeting PI3K and mTOR simultaneously were advantageous to attenuate melanoma growth and they develop their potential by targeting tumor growth directly, and indirectly via their interference with angiogenesis. Based on the above results, NVP-BEZ235, which has entered phase I/II clinical trials in patients with advanced solid tumors, has a potential in metastatic melanoma therapy. (Mol Cancer Res 2009;7(4):601–13)
Increased Expression of Androgen Receptor Coregulator MAGE-11 in Prostate Cancer by DNA Hypomethylation and Cyclic AMP
Authors: Karpf Adam R. , Bai Suxia , James Smitha R. , Mohler James L. , Wilson Elizabeth M.
Published: April 2009

Melanoma antigen gene protein-A11 (MAGE-11) of the MAGE family of cancer germ-line antigens increases androgen receptor (AR) transcriptional activity through its interaction with the AR NH2-terminal FXXLF motif. The present study investigated the regulatory mechanisms that control MAGE-11 expression during androgen deprivation therapy and prostate cancer progression. Studies include the CWR22 xenograft model of human prostate cancer, clinical specimens of benign and malignant prostate, and prostate cancer cell lines. MAGE-11 mRNA levels increased 100- to 1,500-fold during androgen deprivation therapy and prostate cancer progression, with highest levels in the castration-recurrent CWR22 xenograft and clinical specimens of castration-recurrent prostate cancer. Pyrosequencing of genomic DNA from prostate cancer specimens and cell lines indicated the increase in MAGE-11 resulted from DNA hypomethylation of a CpG island in the 5′ promoter of the MAGE-11 gene. Sodium bisulfite sequencing of genomic DNA from benign and malignant prostate tumors and prostate cancer cell lines revealed DNA hypomethylation at individual CpG sites at the transcription start site were most critical for MAGE-11 expression. Cyclic AMP (cAMP) also increased MAGE-11 expression and AR transcriptional activity in prostate cancer cell lines. However, cAMP did not alter DNA methylation of the promoter and its effects were inhibited by extensive DNA methylation in the MAGE-11 promoter region. Increased expression of the AR coregulator MAGE-11 through promoter DNA hypomethylation and cAMP provides a novel mechanism for increased AR signaling in castration-recurrent prostate cancer. (Mol Cancer Res 2009;7(4):523–35)
Loss of p53 and MCT-1 Overexpression Synergistically Promote Chromosome Instability and Tumorigenicity
Authors: Kasiappan Ravi , Shih Hung-Ju , Chu Kang-Lin , Chen Wei-Ti , Liu Hui-Ping , Huang Shiu-Feng , Choy Chik On , Shu Chung-Li , Din Richard , Chu Jan-Show , Hsu Hsin-Ling
Published: April 2009

MCT-1 oncoprotein accelerates p53 degradation by means of the ubiquitin-dependent proteolysis. Our present data show that induction of MCT-1 increases chromosomal translocations and deregulated G2-M checkpoint in response to chemotherapeutic genotoxin. Remarkably, increases in chromosome copy number, multinucleation, and cytokinesis failure are also promoted while MCT-1 is induced in p53-deficient cells. In such a circumstance, the Ras–mitogen-activated protein kinase/extracellular signal-regulated kinase kinase–mitogen-activated protein kinase signaling activity and the expression of metastatic molecules are amplified. Given a p53-silencing background, MCT-1 malignantly transforms normal breast epithelial cells that are satisfactory for stimulating cell migration/adhesion and tumorigenesis. Detailed analyses of MCT-1 oncogenicity in H1299 p53-null lung cancer cells have shown that ectopically expressed MCT-1 advances xenograft tumorigenicity and angiogenesis, which cannot be completely suppressed by induction of p53. MCT-1 counteracts mutually with p53 at transcriptional levels. Clinical validations confirm that MCT-1 mRNA levels are differentially enriched in comparison between human lung cancer and nontumorigenic tissues. The levels of p53 mRNA are comparatively reduced in a subset of cancer specimens, which highly present MCT-1 mRNA. Our results indicate that synergistic promotions of chromosomal imbalances and oncogenic potency as a result of MCT-1 expression and p53 loss play important roles in tumor development. (Mol Cancer Res 2009;7(4):536–48)
Matrix Metalloproteinase–Activated Anthrax Lethal Toxin Inhibits Endothelial Invasion and Neovasculature Formation during In vitro Morphogenesis
Authors: Alfano Randall W. , Leppla Stephen H. , Liu Shihui , Bugge Thomas H. , Meininger Cynthia J. , Lairmore Terry C. , Mulne Arlynn F. , Davis Samuel H. , Duesbery Nicholas S. , Frankel Arthur E.
Published: April 2009

Solid tumor growth is dependent on angiogenesis, the formation of neovasculature from existing vessels. Endothelial activation of the extracellular signal–regulated kinase 1/2, c-jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways is central to this process, and thus presents an attractive target for the development of angiogenesis inhibitors. Anthrax lethal toxin (LeTx) has potent catalytic mitogen-activated protein kinase inhibition activity. Preclinical studies showed that LeTx induced potent tumor growth inhibition via the inhibition of xenograft vascularization. However, LeTx receptors and the essential furin-like activating proteases are expressed in many normal tissues, potentially limiting the specificity of LeTx as an antitumor agent. To circumvent nonspecific LeTx activation and simultaneously enhance tumor vascular targeting, a substrate preferably cleaved by the gelatinases class of matrix metalloproteinases (MMP) was substituted for the furin LeTx activation site. In vivo efficacy studies showed that this MMP-activated LeTx inhibited tumor xenografts growth via the reduced migration of endothelial cells into the tumor parenchyma. Here we have expanded on these initial findings by showing that this MMP-activated LeTx reduces endothelial proangiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, our data suggest that inhibition of the c-jun NH2-terminal kinase and p38, but not extracellular signal–regulated kinase-1/2, pathways is significant in the antiangiogenic activity of the MMP-activated LeTx. Collectively, these results support the clinical development of the MMP-activated LeTx for the treatment of solid tumors. (Mol Cancer Res 2009;7(4):452–61)
Two populations of double minute chromosomes harbor distinct amplicons, the MYC locus at 8q24.2 and a 0.43-Mb region at 14q24.1, in the SW613-S human carcinoma cell line
Authors: Guillaud-Bataille M. , Brison O. , Danglot G. , Lavialle C. , Raynal B. , Lazar V. , Dessen P. , Bernheim A.
Published: April 2009

High-level amplifications observed in tumor cells are usually indicative of genes involved in oncogenesis. We report here a high resolution characterization of a new amplified region in the SW613-S carcinoma cell line. This cell line contains tumorigenic cells displaying high-level MYC amplification in the form of double minutes (DM+ cells) and non tumorigenic cells exhibiting low-level MYC amplification in the form of homogeneously staining regions (DM– cells). Both cell types were studied at genomic and functional levels. The DM+ cells display a second amplification, corresponding to the 14q24.1 region, in a distinct population of DMs. The 0.43-Mb amplified and overexpressed region contains the PLEK2, PIGH, ARG2, VTI1B, RDH11, and ZFYVE26 genes. Both amplicons were stably maintained upon in vitro and in vivo propagation. However, the 14q24.1 amplicon was not found in cells with high-level MYC amplification in the form of HSRs, either obtained after spontaneous integration of endogenous DM MYC copies or after transfection of DM– cells with a MYC gene expression vector. These HSR-bearing cells are highly tumorigenic. The 14q24.1 amplification may not play a role in malignancy per se but might contribute to maintaining the amplification in the form of DMs.
Whole-comparative genomic hybridization in domestic sheep (Ovis aries) breeds
Authors: Dávila-Rodríguez M.I. , Cortés-Gutiérrez E.I. , López-Fernández C. , Pita M. , Mezzanotte R. , Gosálvez J.
Published: April 2009

Whole-comparative genomic hybridization (W-CGH) allows identification of chromosomal polymorphisms related to highly repetitive DNA sequences localized in constitutive heterochromatin. Such polymorphisms are detected establishing competition between genomic DNAs in an in situ hybridization environment without subtraction of highly repetitive DNA sequences, when comparing two species from closely related taxa (same species, sub-species, or breeds) or somewhat related taxa. This experimental approach was applied to investigating differences in highly repetitive sequences of three sheep breeds (Castellana, Ojalada, and Assaf). To this end, W-CGH was carried out using mouflon (sheep ancestor) chromosomes as a common target to co-hybridize equimolar quantities of two genomic DNAs obtained from either Castellana, Ojalada or Assaf sheep breeds. The results showed that the amount of constitutive heterochromatin is greater in all pericentromeric heterochromatin regions of acrocentric chromosomes than in metacentric or sex chromosomes. Additionally, when W-CGH was performed using DNAs from the Iberian breeds Castellana and Ojalada, chromosomal pericentromeric regions revealed quantitatively and qualitatively a presence of DNA families similar to that obtained from any of the above-cited breeds. On the contrary, when the DNA used in W-CGH experiments was obtained from Assaf, as compared to either Castellana or Ojalada, two different pericentromeric DNA families of highly repetitive sequences could be detected. Lastly, sex chromosomes were shown to be homogeneous among all breeds and thus revealed no detectable constitutive heterochromatin. W-CGH results were confirmed using DNA breakage detection-FISH experiments (DBD-FISH) carried out on lymphocytes. As a whole, the results showed that two different repetitive DNA families are present in the pericentromeric heterochromatin of the sheep breeds studied here. Additionally, they suggest a differential presence of these distinct repetitive DNA families in Castellana and Ojalada breeds as compared to the Assaf breed. Finally, the results of W-CGH after using mouflon as the targeted chromosomes also show that the two DNA families are present in the ancestor.
A new pericentromeric repeated DNA sequence in Microtus thomasi
Authors: Acosta M.J. , Marchal J.A. , Mitsainas G.P. , Rovatsos M.T. , Fernández-Espartero C.H. , Giagia-Athanasopoulou E.B. , Sánchez A.
Published: April 2009

Several karyotypic forms have been previously described in populations of the vole species Microtus thomasi from Greece. In particular, the karyomorphs Microtus thomasi ‘thomasi’ and ‘atticus’ differ in X chromosome morphology, being acrocentric and subtelocentric, respectively. Furthermore, remarkable heterochromatin content variability has been described in sex chromosomes of both karyomorphs. Genomic DNA digestion with AluI allowed us to clone an 884 bp long repeated DNA sequence (Mth-Alu900) from the karyomorph M. thomasi ‘atticus’. This repeated DNA is AT rich and seems to be organized mainly as a dimer of the 884-bp unit, which presents three simple repeats (CAAAT, CAGAT and CAGAC) that constitute 80% of the total unit length. This repeated DNA is exclusive to M. thomasi, since it is absent from the genome of other studied Arvicolinae species. The chromosomal location of Mth-Alu900 was analyzed on M. thomasi ‘thomasi’ and M. thomasi ‘atticus’ karyomorphs, with different sex chromosome constitution. It was mainly located on the pericentromeric heterochromatin of most autosomes and X chromosomes on both karyomorphs. Results are also discussed in relation to karyotypic and sex chromosome variations in M. thomasi. To our knowledge, Mth-Alu900 constitutes a new – the third discovered so far – pericentromeric repeated DNA sequence described in Microtus species.
An examination of intergenomic exchanges in A. laterale-dependent unisexual salamanders in the genus Ambystoma
Authors: Bi K. , Bogart J.P. , Fu J.
Published: April 2009

The evolutionary longevity of unisexual salamanders in the genus Ambystoma may be attributed to their flexible reproductive system and meiotic intergenomic interactions. More than 20 different unisexual genomic combinations have been found and all the unisexuals live with at least one of the sexual species A. laterale, A. jeffersonianum, A. texanum, and A. tigrinum. Most unisexuals rely on A. laterale orA. jeffersonianum as sperm donors. Intergenomic exchanges were previously reported in A. jeffersonianum-dependent unisexual populations from southern Ontario and are believed to be an important meiotic mechanism that provides genetic diversity. The situations of intergenomic exchanges in many of A. laterale-dependent unisexual populations, however, remain unknown. In this study we collected specimens from populations where unisexuals use A. laterale as sperm donors, including mainly triploid A. 2 laterale – jeffersonianum (or LLJ), and employed genomic in situ hybridization (GISH) to examine the intergenomic exchanges. Five patterns of intergenomic exchanges were detected. Intergenomic exchanges are less frequent and lack association among populations in A. laterale-dependent than in A. jeffersonianum- dependent unisexual populations, but more recombined homeologues were observed in LLJ unisexuals. Our observations show that the patterns and frequencies of intergenomic exchanges are different when unisexuals use different sexual species as sperm donors. We propose a few possible mechanisms that may account for these different observations.
Characterization of RUSI, a telomere-associated satellite DNA, in the genus Rumex (Polygonaceae)
Authors: Navajas-Pérez R. , Schwarzacher T. , Ruiz Rejón M. , Garrido-Ramos M.A.
Published: April 2009

A satellite-DNA family (RUSI) has been isolated and characterized in Rumexinduratus Boiss and Reuter (Polygonaceae), an Iberian endemic polygamous sorrel. The RUSI repeats are 170 bp in length and ∼68% AT-rich containing different variants of degenerate telomere motifs – (TT)nAN(GG)n –, a typical feature of subtelomeric DNA repeats adjacent to telomeres, which have been referred to as telomere-associated sequences or TASs. In fact, fluorescent in situhybridization showed that this satellite DNA is located in subtelomeric positions of most of the chromosomes of R. induratus, with some centromeric loci. PCR and Southern-blot hybridization assays for sequence conservation in the genus Rumex, indicated that the RUSI sequences are restricted to the genomes of R. induratus and R. scutatus, both species of the section Scutati, suggesting that they are recently evolved. Sequence variation within the two species is high (mean value of sequence differences between repeats of 15% for R. induratus and 7.5% for R. scutatus) and the degree of sequence differentiation between species is low with no species-specific variants, postulated to be due to slowed rates of spreading of sequence variants by molecular homogenizing mechanisms. Characteristics of RUSI sequences are discussed in the light of their chromosomal location and analyzed for their evolutionary and phylogenetic implications.
Rare finding of 2n/4n mixoploidy in mother and fetus with severe immune hydrops
Authors: Sharma A. , Paliwal P. , Dadhwal V. , Sharma Y. , Deka D.
Published: April 2009

The findings of diploid-tetraploid mosaicism in cultured amniotic fluid cells is considered a culture artifact. On rare occasions its presence indicates a chromosomally abnormal fetus with multiple congenital defects. We present here a patient who in her previous pregnancies had delivered one hydrops and two macerated fetuses. The reported pregnancy also resulted in a fetus with severe hydrops. Cytogenetic analysis of cultured amniotic fluid cells revealed diploid-tetraploid mosaicism in the fetus also confirmed in fetal cord blood and skin fibroblasts. Chromosomal analysis of the parents revealed mixoploidy in the mother. These findings are extremely important for prenatal diagnosis and prompt us to not uniformly dismiss tetraploidy as artifactual but to confirm it especially in cases with ultrasound abnormalities.
FISH-mapping comparison between river buffalo chromosome 7 and sheep chromosome 6: assignment of new loci and comparison with HSA4
Authors: Perucatti A. , Di Meo G.P. , Goldammer T. , Incarnato D. , Nicolae I. , Brunner R. , Iannuzzi L.
Published: April 2009

Synchronized peripheral blood lymphocytes from both river buffalo (BBU) and sheep (OAR) were treated for late incorporation of both BrdU and H-33258 to obtain R-banded preparations to be used for FISH-mapping. Ovine BAC-clones were hybridized for three days on slides pre-exposed to UV light after H-33258 staining. The following loci were mapped: GPR103 (BBU7q13, OAR6q13), TRAM1L1(OAR6q13dist), PPP3CA (BBU7q21, OAR6q15), SNCA (OAR6q17), PPARGC1A(BBU7q23, OAR6q17), UGDH (BBU7q25prox, OAR6q22prox), KDR (BBU7q27, OAR6q22), CNOT6L (BBU7q32prox, OAR6q32prox), NUP54 (BBU7q32, BBU6q32), DMP1 (BBU7q34dist-q36prox, OAR6q34dist-q36prox), QDPR (BBU7q36, OAR6q36). All loci mapped in homoeologous chromosomes and chromosome bands of the two species and their locations are in agreement with the previous RH-mapping performed on BBU7 with some difference in the distal region of BBU7. However, the present cytogenetic map better anchors the RH-map on specific river buffalo chromosome bands. In addition, eleven loci were assigned for the first time in sheep to OAR6, noticeably extending the cytogenetic map on this important chromosome which encodes caseins. Two loci (TRAM1L1 and SNCA) mapped in sheep were unmapped in river buffalo in three different FISH experiments. Comparisons between integrated cytogenetic maps of BBU7/OAR6 (and BTA6) with human chromosome 4 (HSA4) revealed complex chromosome rearrangements differentiating these chromosomes.
Skin Cancer: New Markers for Better Prevention
Authors: Greinert Rüdiger
Published: April 2009

Skin cancer is the most frequent cancer in the white population worldwide. Incidence of basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma (MM) is still increasing. This trend can be counteracted by means of primary and secondary prevention because the main risk factor for skin cancer – UV-radiation – is known, and, early detected, skin cancer can be cured successfully. For early detection of skin cancer suitable risk (group) markers have to be used to identify persons at risk. In order to increase the sensitivity and specificity of early detection efforts (screening programs) new molecular markers or biomarkers should be used in the future in the field of molecular epidemiology. In this review the skin cancer problem is summarized and the possible use of new biomarkers for skin cancer development, progression, metastasis and prognosis is discussed. The review focuses on results of gene expression profiling using array techniques and the new possibilities for the use of epigenetic biomarkers.
Cervical Cancer Control in the Era of HPV Vaccination and Novel Biomarkers
Authors: Wentzensen Nicolas , Klug Stefanie J.
Published: April 2009

Infections with human papillomaviruses (HPV) are a necessary, but not sufficient cause of cervical cancer. Cervical cancer develops over a long time through precursor lesions that can be detected by cytological screening. The majority of these lesions regress spontaneously without treatment. The challenge of cervical cancer screening is to detect the lesions that have a high risk of progression. Since the recently introduced vaccination against HPV cannot provide 100% protection, cervical cancer screening programs must continue. It is assumed that the reduction of precancers related to vaccination will have a negative impact on the efficiency of current screening programs. Therefore, participation rates need to be increased and current screening modalities should be improved. Several promising biomarkers have been described that might improve cervical cancer screening, but currently, high-quality studies on their efficiency are lacking.
Physical Principles of Microbubbles for Ultrasound Imaging and Therapy
Authors: Stride Eleanor
Published: April 2009

Microbubble ultrasound contrast agents have been in clinical use for more than two decades, during which time their range of applications has increased to encompass echocardiography, Doppler enhancement, perfusion studies and molecular imaging, as well as a number of therapeutic applications including drug delivery, gene therapy, high-intensity focused ultrasound treatments and sonothrombolysis. The aim of this article is to review the different types of microbubble agent, their physical behaviour and the mechanisms underlying their effectiveness in imaging and therapeutic applications.