bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of an Involvement of the Human Mismatch Repair Genes
hMLH1
and
hMSH2
in Nucleotide Excision Repair Is Dependent on UVC Fluence to Cells
Authors: Lee David F. , Drouin Regen , Pitsikas Photini , Rainbow Andrew J.
Published: September 2005

There is conflicting evidence for the role of the mismatch repair (MMR) genes hMLH1 and hMSH2 in the transcription-coupled repair (TCR) pathway of nucleotide excision repair. In the present work, we have examined the role of these MMR genes in nucleotide excision repair using two reporter gene assays. AdHCMVlacZ is a replication-deficient recombinant adenovirus that expresses the β-galactosidase reporter gene under the control of the human cytomegalovirus immediate early promoter. We have reported previously a reduced host cell reactivation (HCR) for β-galactosidase expression of UVC-irradiated AdHCMVlacZ in TCR-deficient Cockayne syndrome (CS) fibroblasts compared with normal fibroblasts, indicating that HCR depends, at least in part, on TCR. In addition, we have reported that UVC-enhanced expression of the undamaged reporter gene is induced at lower UVC fluences to cells and at higher levels after low UVC fluences in TCR-deficient compared with normal human fibroblasts, suggesting that persistent damage in active genes triggers increased activity from the human cytomegalovirus-driven reporter construct. We have examined HCR and UV-enhanced expression of the reporter gene in hMLH1-deficient HCT116 human colon adenocarcinoma cells and HCT116-chr3 cells (the MMR-proficient counterpart of HCT116) as well as hMSH2-deficient LoVo human colon adenocarcinoma cells and their hMSH2-proficient counterpart SW480 cells. We show a greater UV-enhanced expression of the undamaged reporter gene after low UVC exposure in HCT116 compared with HCT116-chr3 cells and in LoVo compared with SW480 cells. We show also a reduced HCR in HCT116 compared with HCT116-chr3 cells and in LoVo compared with SW480 cells. However, the reduction in HCR was less or absent when cells were pretreated with UVC. These results suggest that detection of an involvement of hMLH1 and hMSH2 in TCR is dependent on UVC (254 nm) fluence to cells.
ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation
Authors: Stiff Tom , O’Driscoll Mark , Rief Nicole , Iwabuchi Kuniyoshi , Löbrich Markus , Jeggo Penny A.
Published: September 2005

H2AX phosphorylation is an early step in the response to DNA damage. It is widely accepted that ATM (ataxia telangiectasia mutated protein) phosphorylates H2AX in response to DNA double-strand breaks (DSBs). Whether DNA-dependent protein kinase (DNA-PK) plays any role in this response is unclear. Here, we show that H2AX phosphorylation after exposure to ionizing radiation (IR) occurs to similar extents in human fibroblasts and in mouse embryo fibroblasts lacking either DNA-PK or ATM but is ablated in ATM-deficient cells treated with LY294002, a drug that specifically inhibits DNA-PK. Additionally, we show that inactivation of both DNA-PK and ATM is required to ablate IR-induced H2AX phosphorylation in chicken cells. We confirm that H2AX phosphorylation induced by DSBs in nonreplicating cells is ATR (ataxia telangiectasia and Rad3-related protein) independent. Taken together, we conclude that under most normal growth conditions, IR-induced H2AX phosphorylation can be carried out by ATM and DNA-PK in a redundant, overlapping manner. In contrast, DNA-PK cannot phosphorylate other proteins involved in the checkpoint response, including chromatin-associated Rad17. However, by phosphorylating H2AX, DNA-PK can contribute to the presence of the damage response proteins MDC1 and 53BP1 at the site of the DSB.
Acquisition of High-Level Chromosomal Instability Is Associated with Integration of Human Papillomavirus Type 16 in Cervical Keratinocytes
Authors: Pett Mark R. , Alazawi William O. F. , Roberts Ian , Dowen Sally , Smith David I. , Stanley Margaret A. , Coleman Nicholas
Published: September 2005

Whereas two key steps in cervical carcinogenesis are integration of high-risk human papillomavirus (HR-HPV) and acquisition of an unstable host genome, the temporal association between these events is poorly understood. Chromosomal instability is induced when HR-HPV E7 oncoprotein is overexpressed from heterologous promoters in vitro. However, it is not known whether such events occur at the “physiologically” elevated levels of E7 produced by deregulation of the homologous HR-HPV promoter after integration. Indeed, an alternative possibility is that integration in vivo is favored in an already unstable host genome. We have addressed these issues using the unique human papillomavirus (HPV) 16-containing cervical keratinocyte cell line W12, which was derived from a low-grade squamous intraepithelial lesion and thus acquired HPV16 by “natural” infection. Whereas W12 at low passage contains HPV16 episomes only, long-term culture results in the emergence of cells containing integrated HPV16 only. We show that integration of HPV16 in W12 is associated with 3′ deletion of the E2 transcriptional repressor, resulting in deregulation of the homologous promoter of the integrant and an increase in E7 protein levels. We further demonstrate that high-level chromosomal instability develops in W12 only after integration and that the forms of instability observed correlate with the physical state of HPV16 DNA and the level of E7 protein. Whereas intermediate E7 levels are associated with numerical chromosomal abnormalities, maximal levels are associated with both numerical and structural aberrations. HR-HPV integration is likely to be a critical event in cervical carcinogenesis, preceding the development of chromosomal abnormalities that drive malignant progression.
Bcl-2-Dependent Modulation of Swelling-Activated Cl− Current and ClC-3 Expression in Human Prostate Cancer Epithelial Cells
Authors: Lemonnier Loïc , Shuba Yaroslav , Crepin Alexandre , Roudbaraki Morad , Slomianny Christian , Mauroy Brigitte , Nilius Bernd , Prevarskaya Natalia , Skryma Roman
Published: September 2005

Cell shrinkage is an integral part of apoptosis. However, intimate mechanisms linking apoptotic events to the alterations in cell volume homeostasis remain poorly elucidated. We investigated how overexpression of Bcl-2 oncoprotein, a key antiapoptotic regulator, in lymph node carcinoma of the prostate (LNCaP) prostate cancer epithelial cells interferes with the volume-regulated anion channel (VRAC), a major determinant of regulatory volume decrease. Bcl-2 overexpression resulted in the doubling of VRAC-carried swelling-activated Cl− current (ICl,swell) and weakened ICl,swell inhibition by store-operated Ca2+ channel (SOC)-transported Ca2+. This also was accompanied by substantial up-regulation of ClC-3 protein, a putative molecular candidate for the role of VRAC. ClC-3-specific antibody suppressed ICl,swell in the wild-type and Bcl-2-overexpressing LNCaP cells. Epidermal growth factor treatment of wild-type LNCaP cells, promoting their proliferation, resulted in the enhancement of endogenous Bcl-2 expression and associated increases in ClC-3 levels and ICl,swell magnitude. We conclude that Bcl-2-induced up-regulation of ICl,swell, caused by enhanced expression of ClC-3 and weaker negative control from SOC-transported Ca2+, would strengthen the ability of the cells to handle proliferative volume increases and thereby promote their survival and diminish their proapoptotic potential.
CD4+ T-Cell Recognition of Mutated B-RAF in Melanoma Patients Harboring the V599E Mutation
Authors: Sharkey Melinda S. , Lizée Gregory , Gonzales Monica I. , Patel Sima , Topalian Suzanne L.
Published: September 2005

The potential of antigen-directed cancer immunotherapy has not been fully realized, perhaps because many commonly targeted tumor associated proteins are not essential to maintaining the malignant cell phenotype. A constitutively activating mutation in the signaling molecule BRAF is expressed frequently in melanomas and may play an important role in the biology of this disease. A 29-mer B-Raf peptide incorporating the V599E mutation was used for in vitro stimulation of lymphocytes derived from melanoma patients, generating MHC class II-restricted CD4+ T cells specific for this peptide as well as for melanoma cells expressing B-Raf V599E. Mutated B-Raf exemplifies targets that may be ideal for immunotherapy.
Tobacco Carcinogen-Induced Cellular Transformation Increases Activation of the Phosphatidylinositol 3′-Kinase/Akt Pathwayin Vitroandin Vivo
Authors: West Kip A. , Linnoila Ilona R. , Belinsky Steven A. , Harris Curtis C. , Dennis Phillip A.
Published: September 2005

The role of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway during tobacco carcinogen-induced transformation is unknown. To address this question, we evaluated this pathway in isogenic immortalized or tumorigenic human bronchial epithelial cells in vitro, as well as in progressive murine lung lesions induced by a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Compared with immortalized cells, tumorigenic cells had greater activation of the PI3K/Akt pathway, enhanced survival, and increased apoptosis in response to inhibition of the pathway. In vivo, increased activation of Akt and mammalian target of rapamycin was observed with increased phenotypic progression. Collectively, these results support the hypothesis that maintenance of Akt activity is necessary for survival of preneoplastic as well as transformed lung epithelial cells and suggest that inhibition of the PI3K/Akt pathway might be a useful approach to arrest lung tumorigenesis.
Early Occurrence of
RASSF1A
Hypermethylation and Its Mutual Exclusion with
BRAF
Mutation in Thyroid Tumorigenesis
Authors: Xing Mingzhao , Cohen Yoram , Mambo Elizabeth , Tallini Giovanni , Udelsman Robert , Ladenson Paul W. , Sidransky David
Published: September 2005

Follicular epithelial cell-derived thyroid tumors are common neoplasms comprised mainly of benign thyroid adenomas, follicular thyroid cancers, and papillary thyroid cancers (PTCs). Hypermethylation of the tumor suppressor gene RASSF1A and activating mutation of BRAF gene have been reported recently in thyroid cancers. To additionally investigate the roles of these two epigenetic/genetic alterations in thyroid tumor progression, we examined their occurrences and relationship in both benign and malignant thyroid neoplasms. With real-time quantitative methylation-specific PCR, we found that 4 of 9 (44%) benign adenomas, 9 of 12 (75%) follicular thyroid cancers tumors, and 6 of 30 (20%) of PTC tumors harbored promoter methylation in ≥25% of RASSF1A alleles. Additional quantitative analysis revealed RASSF1A methylation only in BRAF mutation-negative PTCs. A similar inverse correlation of RASSF1A methylation with BRAF mutation was seen in thyroid tumor cell lines. Our results, therefore, suggest that epigenetic inactivation of RASSF1A through aberrant methylation is an early step in thyroid tumorigenesis. Like the previously reported mutually exclusive relationship between BRAF mutation and other Ras pathway components such as RET/PTC rearrangement, a mutually exclusive relationship also exists between BRAF mutation and RASSF1A methylation in thyroid tumorigenesis.
Local Administration of Granulocyte/Macrophage Colony-stimulating Factor Increases the Number and Activation State of Dendritic Cells in the Sentinel Lymph Node of Early-Stage Melanoma
Authors: Vuylsteke Ronald J. C. L. M. , Molenkamp Barbara G. , Gietema Hester A. , van Leeuwen Paul A. M. , Wijnands Pepijn G. J. T. B. , Vos Wim , van Diest Paul J. , Scheper Rik J. , Meijer Sybren , de Gruijl Tanja D.
Published: September 2005

The initial tumor-draining lymph node, the sentinel lymph node, not only constitutes the first expected site of micrometastasis but also the first point of contact between tumor-associated antigens and the adaptive immune system. A tumor-induced decrease in the frequency and activation state of sentinel lymph node dendritic cells will impair the generation of effective antitumor T-cell responses and increase the likelihood of metastatic spread. Here, we demonstrate that intradermal administration of granulocyte macrophage-colony stimulating factor around the excision site of stage I primary melanoma tumors increases the number and activation state of dendritic cells in the paracortical areas of the sentinel lymph node and enhances their binding to T cells. We conclude that local treatment of melanoma patients with granulocyte macrophage-colony stimulating factor, before surgery, conditions the sentinel lymph node microenvironment to enhance mature dendritic cell recruitment and hypothesize that this may be more conducive to the generation of T-cell–mediated antitumor immunity.
PD-L1/B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor (TCR) Transgenic CD8+ T Cells
Authors: Blank Christian , Brown Ian , Peterson Amy C. , Spiotto Mike , Iwai Yoshiko , Honjo Tasuku , Gajewski Thomas F.
Published: September 2005

Although increased circulating tumor antigen-specific CD8+ T cells can be achieved by vaccination or adoptive transfer, tumor progression nonetheless often occurs through resistance to effector function. To develop a model for identifying mechanisms of resistance to antigen-specific CTLs, poorly immunogenic B16-F10 melanoma was transduced to express the Kb-binding peptide SIYRYYGL as a green fluorescent protein fusion protein that should be recognized by high-affinity 2C TCR transgenic T cells. Although B16.SIY cells expressed high levels of antigen and were induced to express Kb in response to IFN-γ, they were poorly recognized by primed 2C/RAG2−/− T cells. A screen for candidate inhibitory ligands revealed elevated PD-L1/B7H-1 on IFN-γ-treated B16-F10 cells and also on eight additional mouse tumors and seven human melanoma cell lines. Primed 2C/RAG2−/−/PD-1−/− T cells showed augmented cytokine production, proliferation, and cytolytic activity against tumor cells compared with wild-type 2C cells. This effect was reproduced with anti-PD-L1 antibody present during the effector phase but not during the priming culture. Adoptive transfer of 2C/RAG2−/−/PD-1−/− T cells in vivo caused tumor rejection under conditions in which wild-type 2C cells or CTLA-4-deficient 2C cells did not reject. Our results support interfering with PD-L1/PD-1 interactions to augment the effector function of tumor antigen-specific CD8+ T cells in the tumor microenvironment.
A Chemically Induced Model for Squamous Cell Carcinoma of the Lung in Mice
Authors: Wang Yian , Zhang Zhongqiu , Yan Ying , Lemon William J. , LaRegina Marie , Morrison Carl , Lubet Ronald , You Ming
Published: September 2005

Lung cancer, primarily associated with tobacco use, is the leading cause of cancer morbidity and mortality in the United States. Squamous cell carcinoma (SCC) is one of the four major histological types of lung cancer. Although there are several established models for lung adenoma and adenocarcinomas, there is no well-established mouse model for lung SCC. We treated eight different inbred strains of mice with N-nitroso-tris-chloroethylurea by skin painting and found that this regimen induced lung SCCs in five strains of mouse (SWR/J, NIH Swiss, A/J, BALB/cJ, and FVB/J) but not in the others (AKR/J, 129/svJ, and C57BL/6J). Mouse lung SCCs have similar histopathological features and keratin staining to human SCC. Moreover, a wide spectrum of abnormal lung squamous phenotypes including hyperplasia, metaplasia, carcinoma in situ, and invasive carcinoma, were observed. There are strain-specific differences in susceptibility to Lscc induction by N-nitroso-tris-chloroethylurea with NIH Swiss, A/J, and SWR/J mice developing scores of SCCs whereas the resistant strains AKR/J, 129/svJ, and C57BL/6J failed to develop any SCCs. FVB/J and BALB/cJ mice had an intermediate response. We conducted whole-genome linkage disequilibrium analysis in seven strains of mice, divided into three phenotype categories of susceptibility, using Fisher’s exact test applied to 6,128 markers in publically available databases. Three markers were found significantly associated with susceptibility to SCC with the P < 0.05. They were D1Mit169, D3Mit178, and D18Mit91. Interestingly, none of these sites overlap with the major susceptibility loci associated with lung adenoma/adenocarcinoma development in mice. The mouse SCC described here is highly significant for preclinical studies of lung cancer chemopreventive agents because most human trials have been conducted against precancerous lesions for SCC. Furthermore, this model can be used in determining genetic modifiers that contribute to susceptibility or resistance to lung SCC development.
Sequence Variants ofToll-Like Receptor 4Are Associated with Prostate Cancer Risk
Authors: Zheng S. Lilly , Augustsson-Bälter Katarina , Chang Baoli , Hedelin Maria , Li Liwu , Adami Hans-Olov , Bensen Jeanette , Li Ge , Johnasson Jan-Erik , Turner Aubrey R. , Adams Tamara S. , Meyers Deborah A. , Isaacs William B. , Xu Jianfeng , Grönberg Henrik
Published: September 2005

Inflammation has been implicated as an etiological factor in several human cancers. Growing evidence suggests that chronic inflammation may also play a role in the etiology of prostate cancer. Considering that genetic susceptibility is a major risk factor for this disease, we hypothesize that sequence variants in genes that regulate inflammation may modify individual susceptibility to prostate cancer. The lipopolysaccharide receptor Toll-like receptor 4 (TLR4) is a central player in the signaling pathways of the innate immune response to infection by Gram-negative bacteria and is an important candidate inflammatory gene. We performed a systematic genetic analysis of TLR4 sequence variants by evaluating eight single-nucleotide polymorphisms that span the entire gene among 1383 newly diagnosed prostate cancer patients and 780 age- and residence-matched controls in Sweden. We found an association between a sequence variant (11381G/C) in the 3′-untranslated region of the TLR4 gene and prostate cancer risk. The frequency of the variant genotypes (CG or CC) was significantly higher in the patients (24.1%) than in the controls (19.7%; P = 0.02). The frequency of risk genotypes among patients diagnosed before the age of 65 years was even higher (26.3%). Compared with men who had the wild-type genotype of this single-nucleotide polymorphism (GG), those with GC or CC genotypes had a 26% increased risk for prostate cancer (odds ratio, 1.26; 95% confidence interval, 1.01–1.57) and 39% increased risk increased risk for early onset prostate cancer (before age 65 years; odds ratio, 1.39; 95% confidence interval, 1.02–1.91). The risk attributable to this variant for prostate cancer in Sweden was estimated to be 4.9%. Although the biological mechanism of the observed association remains to be elucidated, our finding supports a role for a bacteria-associated response pathway, possibly acting via inflammation, in the development of prostate cancer.
2-Deoxy-d-glucose Increases the Efficacy of Adriamycin and Paclitaxel in Human Osteosarcoma and Non-Small Cell Lung Cancers
In Vivo
Authors: Maschek Gregory , Savaraj Niramol , Priebe Waldemar , Braunschweiger Paul , Hamilton Kara , Tidmarsh George F. , De Young Linda R. , Lampidis Theodore J.
Published: September 2005

Slow-growing cell populations located within solid tumors are difficult to target selectively because most cells in normal tissues also have low replication rates. However, a distinguishing feature between slow-growing normal and tumor cells is the hypoxic microenvironment of the latter, which makes them extraordinarily dependent on anaerobic glycolysis for survival. Previously, we have shown that hypoxic tumor cells exhibit increased sensitivity to inhibitors of glycolysis in three distinct in vitro models. Based on these results, we predicted that combination therapy of a chemotherapeutic agent to target rapidly dividing cells and a glycolytic inhibitor to target slow-growing tumor cells would have better efficacy than either agent alone. Here, we test this strategy in vivo using the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) in combination with Adriamycin (ADR) or paclitaxel in nude mouse xenograft models of human osteosarcoma and non-small cell lung cancer. Nude mice implanted with osteosarcoma cells were divided into four groups as follows: (a) untreated controls; (b) mice treated with ADR alone; (c) mice treated with 2-DG alone; or (d) mice treated with a combination of ADR + 2-DG. Treatment began when tumors were either 50 or 300 mm3 in volume. Starting with small or large tumors, the ADR + 2-DG combination treatment resulted in significantly slower tumor growth (and therefore longer survival) than the control, 2-DG, or ADR treatments (P < 0.0001). Similar beneficial effects of combination treatment were found with 2-DG and paclitaxel in the MV522 non-small cell lung cancer xenograft model. In summary, the treatment of tumors with both the glycolytic inhibitor 2-DG and ADR or paclitaxel results in a significant reduction in tumor growth compared with either agent alone. Overall, these results, combined with our in vitro data, provide a rationale for initiating clinical trials using glycolytic inhibitors in combination with chemotherapeutic agents to increase their therapeutic effectiveness.
The Functional
UGT1A1
Promoter Polymorphism Decreases Endometrial Cancer Risk
Authors: Duguay Yannick , McGrath Monica , Lépine Johanie , Gagné Jean-François , Hankinson Susan E. , Colditz Graham A. , Hunter David J. , Plante Marie , Têtu Bernard , Bélanger Alain , Guillemette Chantal , De Vivo Immaculata
Published: September 2005

UDP-glucuronosyltransferase (UGT) 1A1 is involved in the inactivation of estradiol (E2) and its oxidized metabolites. These metabolites have been shown to contribute to the development of endometrial cancer in animal studies. Thus UGT1A1 represents a candidate gene in endometrial carcinogenesis. In this study, we established the substrate specificity of UGT1A1 for E2 and its 2- and 4-hydroxylated metabolites. Intrinsic clearances indicated that UGT1A1 had a preference for the glucuronidation of 2-hydroxyestradiol, a metabolite associated with antiproliferative activity. Expression analysis demonstrated that UGT1A1 is present in the nonmalignant endometrium. Subsequently, we sought to determine whether the common UGT1A1 promoter allele, UGT1A1*28 [A(TA)7TAA], which decreases gene transcription, was associated with endometrial cancer risk in a case-control study nested within the Nurses’ Health Study (222 cases, 666 matched controls). Conditional logistic regression demonstrated a significant inverse association with the UGT1A1*28 allele and endometrial cancer risk. Compared with women homozygous for the UGT1A1*1 [A(TA)6TAA] allele, the adjusted odds ratio (OR) was 0.81 [95% confidence interval (CI), 0.56–1.16] for the UGT1A1*1/*28 genotype and 0.40 (95% CI, 0.21–0.75) for the homozygous UGT1A1*28 genotype (Ptrend = 0.007). There was a suggestion of an interaction by menopausal status [OR = 0.39 (95% CI, 0.18–0.85) for premenopausal women and OR = 0.79 (95% CI, 0.55–1.13) for postmenopausal women who carry the UGT1A1*28 allele (Pinteraction = 0.05)]. These observations suggest that lower expression of UGT1A1 decreases the risk of endometrial cancer by reducing the excretion of 2-hydroxyestradiol, the antiproliferative metabolite of E2, in the endometrium.
Cooperative Autocrine and Paracrine Functions of Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor in the Progression of Skin Carcinoma Cells
Authors: Obermueller Eva , Vosseler Silvia , Fusenig Norbert E. , Mueller Margareta M.
Published: September 2005

Tumor growth and progression are critically controlled by alterations in the microenvironment often caused by an aberrant expression of growth factors and receptors. We demonstrated previously that tumor progression in patients and in the experimental HaCaT tumor model for skin squamous cell carcinomas is associated with a constitutive neoexpression of the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), causing an autocrine stimulation of tumor cell proliferation and migration in vitro. To analyze the critical contribution of both factors to tumor progression, G-CSF or GM-CSF was stably transfected in factor-negative benign tumor cells. Forced expression of GM-CSF resulted in invasive growth and enhanced tumor cell proliferation in a three-dimensional culture model in vitro, yet tumor growth in vivo remained only transient. Constitutive expression of G-CSF, however, caused a shift from benign to malignant and strongly angiogenic tumors. Moreover, cells recultured from G-CSF–transfected tumors exhibited enhanced tumor aggressiveness upon reinjection, i.e., earlier onset and faster tumor expansion. Remarkably, this further step in tumor progression was again associated with the constitutive expression of GM-CSF strongly indicating a synergistic action of both factors. Additionally, expression of GM-CSF in the transfected tumors mediated an earlier recruitment of granulocytes and macrophages to the tumor site, and expression of G-CSF induced an enhanced and persistent angiogenesis and increased the number of granulocytes and macrophages in the tumor vicinity. Thus both factors directly stimulate tumor cell growth and, by modulating the tumor stroma, induce a microenvironment that promotes tumor progression.
The Potential Role of Hypoxia Inducible Factor 1α in Tumor Progression after Hypoxia and Chemotherapy in Hepatocellular Carcinoma
Authors: Yang Zhen Fan , Poon Ronnie T. , To Jensen , Ho David W. , Fan Sheung Tat
Published: September 2005

This study investigates the possible molecular basis leading to failure in a treatment that is composed of hypoxia and chemotherapy in a rat orthotopic hepatoma model. Hypoxia was induced by hepatic artery ligation, whereas chemotherapeutic effect was achieved by intraportal injection of cisplatin. High-dose sodium salicylate was administered to achieve transcriptional blockade. Significant prolongation of animal survival was observed in the groups receiving hepatic artery ligation with cisplatin or sodium salicylate. Massive tumor cell necrosis and apoptosis were found in the ligation and all of the combined treatment groups. Up-regulation of hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) at both mRNA and protein levels were detected in the groups receiving ligation and ligation with cisplatin, whereas a decreased level of von Hippel-Lindau tumor suppressor protein was identified in the group receiving ligation with cisplatin. Sodium salicylate enhanced expression of von Hippel-Lindau tumor suppressor protein but down-regulated HIF-1α and VEGF levels after ligation with or without cisplatin. An increased number of activated hepatic stellate cells in the tumors were observed in the ligation and ligation with cisplatin groups, whereas they were greatly reduced by sodium salicylate. In vitro study revealed that under hypoxic condition, both cisplatin and sodium salicylate could remarkably augment P53 and caspase 3 levels. Cisplatin stimulated HIF-1α up-regulation, whereas sodium salicylate suppressed HIF-1α expression. In conclusion, tumor progression after hypoxia and chemotherapy might be related to up-regulation of HIF-1α and subsequent VEGF production, and transcriptional blockade by sodium salicylate could enhance the therapeutic efficacy of hypoxia and chemotherapy.
Apoptotic Speck Protein-Like, a Highly Homologous Protein to Apoptotic Speck Protein in the Pyrin Domain, Is Silenced by DNA Methylation and Induces Apoptosis in Human Hepatocellular Carcinoma
Authors: Kubo Takahiko , Yamamoto Junji , Shikauchi Yuko , Niwa Yasuharu , Matsubara Kenichi , Yoshikawa Hirohide
Published: September 2005

We have identified a novel gene encoding a pyrin domain protein of 89 amino acids that is expressed in various tissues including liver, brain, and spleen. The protein is highly homologous to the pyrin domain of apoptosis-associated speck-like protein (ASC). Therefore, we termed it ASC-like (ASCL). We found that ASCL gene was densely and frequently (80%) methylated in hepatocellular carcinoma (HCC) cell lines. In contrast, normal liver samples did not show any significant methylation. This aberrant methylation correlated well with the suppression of RNA expression. Furthermore, a demethylating agent, 5-aza-2′-deoxycytidine, reactivated the ASCL expression in the methylation-silenced cells, indicating that ASCL is silenced by the associated DNA methylation. ASCL methylation was also found in primary HCC (4 of 17 samples), although the frequency was less than that in cell lines. In addition, we found that ASC was also methylated in primary samples (6 of the 17). Interestingly, either ASCL or ASC methylation was observed in 53% (9 of the 17) of primary HCC samples. Significantly, the restoration of ASCL in the methylation-silenced cells demonstrated growth suppression in colony formation assay. This growth suppression effect of ASCL was supported by apoptotic changes observed in ASCL-transfected cells in which annexin-V binding was positive and caspase-3 was activated. Based on the methylation-silencing and the growth suppression activity, we propose that ASCL plays a significant role in the development of HCC.
Detection of Cyclin B1 Expression in G1-Phase Cancer Cell Lines and Cancer Tissues by Postsorting Western Blot Analysis
Authors: Shen Manli , Feng Yongdong , Gao Chun , Tao Deding , Hu Junbo , Reed Eddie , Li Qingdi Q. , Gong Jianping
Published: September 2005

Protein complex of cyclin B1 and cyclin-dependent protein kinase 1 induces phosphorylation of key substrates that mediate cell cycle transition during the G2-M phase. It is believed that cyclin B1 accumulates in the S phase of the cell cycle and reaches the maximal level at mitosis but is absent in G1-phase cells. In the present study, we demonstrated that cyclin B1 was expressed in the arrested G1-phase MOLT-4 lymphocyte leukemia cells and in G1 phase T-7 transitional tumor cells, as determined by flow cytometry. In addition, we showed that cyclin B1 was detected in the G1 phase in breast cancer cells from patient tissues and in lymphocytes from patients with leukemia. These findings were confirmed for the first time by postsorting Western blot analysis and by confocal microscopy. Furthermore, by using postsorting Western blotting, we found that cyclin B1 was expressed in different time-window sections of the G1 phase under different conditions. For the asynchronously growing T-7 cells, cyclin B1 was detected in early G1 phase, whereas in MOLT-4 cells arrested in G1-S phase, cyclin B1 was mainly detected in late G1 phase. We propose that the cyclin B1 expressed in the G1 phase may differ from that expressed in the G2-M phase, and that this unscheduled type of cyclin B1 may play an important role in tumorigenesis and apoptosis.
Topical Treatment with Inhibitors of the Phosphatidylinositol 3′-Kinase/Akt and Raf/Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Pathways Reduces Melanoma Development in Severe Combined Immunodeficient Mice
Authors: Bedogni Barbara , O’Neill Melony S. , Welford Scott M. , Bouley Donna M. , Giaccia Amato J. , Denko Nicholas C. , Powell Marianne Broome
Published: September 2005

Topical treatment with inhibitors of the phosphatidylinositol 3′-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways inhibited the growth of TPras transgenic melanomas in severe combined immunodeficient mice, blocked invasive behavior, and reduced angiogenesis. The inhibitor Ly294002, which is specific for phosphatidylinositol 3′-kinase, effectively reduced melanoma cell growth both in vitro and in vivo. Both Ly294002 and U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, reduced invasion, which correlated with reduction of the metalloproteinase matrix metalloproteinase 2. Tumor angiogenesis was disrupted through inhibition of vascular endothelial growth factor production from the tumor cells and antiangiogenic effects on endothelial cells. Observations with TPras melanoma cells that express dominant negative Δp85 or kinase-inactive Raf301 supported the specificity of the phenomena observed with the chemical inhibitors. These studies demonstrate that topical treatment targeting Ras effectors is efficacious, without systemic toxicities, and may prove to be useful in treating and preventing the progression of cutaneous melanoma.
Quantitative Multiplex Methylation-Specific PCR Assay for the Detection of Promoter Hypermethylation in Multiple Genes in Breast Cancer
Authors: Fackler Mary Jo , McVeigh Megan , Mehrotra Jyoti , Blum Marissa A. , Lange Julie , Lapides Amanda , Garrett Elizabeth , Argani Pedram , Sukumar Saraswati
Published: September 2005

If detected early, breast cancer is eminently curable. To detect breast cancer in samples with little cellularity, a high level of sensitivity is needed. Tumor-specific promoter hypermethylation has provided such a valuable tool for detection of cancer cells in biological samples. To accurately assess promoter hypermethylation for many genes simultaneously in small samples, we developed a novel method, quantitative multiplex-methylation-specific PCR (QM-MSP). QM-MSP is highly sensitive (1 in 104–105 copies of DNA) and linear over 5 orders of magnitude. For RASSF1A, TWIST, Cyclin D2, and HIN1, we observed significant differences in both the degree (P < 0.003) and incidence (P < 0.02) of hypermethylation between normal and malignant breast tissues. Evaluation of the cumulative hypermethylation of the four genes within each sample revealed a high level of sensitivity (84%) and specificity (89%) of detection of methylation. We demonstrate the application of this technique for detecting hypermethylated RASSF1A, TWIST, Cyclin D2, HIN1, and RARB in 50–1000 epithelial cells collected from breast ducts during endoscopy or by lavage. Such an approach could be used in a variety of small samples derived from different tissues, with these or different biomarkers to enhance detection of malignancy.
Integrin β3 Overexpression Suppresses Tumor Growth in a Human Model of Gliomagenesis
Authors: Kanamori Masayuki , Berg Scott R. Vanden , Bergers Gabriele , Berger Mitchel S. , Pieper Russell O.
Published: September 2005

αVβ3 integrin complexes are overexpressed in the growing, invading margins of human glioblastoma multiforme (GBM) and in the GBM vasculature, suggesting a key role for αVβ3 in GBM growth and invasion. The function of αVβ3 complexes in tumor formation, however, has been challenged by studies showing that loss of αVβ3 expression (via loss of β3) in the host vasculature enhances, rather than suppresses, the growth of s.c. implanted carcinomas. To directly address the role of tumor-specific αVβ3 overexpression in glioma formation, we increased αVβ3 expression (via overexpression of a wild-type or constitutively activated β3) in human astrocytes genetically modified to form anaplastic astrocytoma-like tumors (Ras cells) on intracranial injection in rats. Overexpression of β3 selectively increased levels of αVβ3 integrin complexes, but had no effect on anchorage-dependent or -independent growth in vitro. After intracranial injection, however, the Ras + β3 cells formed fewer and smaller tumors than did Ras cells. Similarly, Ras-transformed mouse astrocytes that were derived from control animals formed smaller intracranial tumors than those derived from β3 knockout animals. Although tumors formed by human Ras and Ras + β3 cells were similar in blood vessel density, Ras + β3 tumors had smaller, pericyte-depleted vessels and were significantly more hypoxic, suggesting a β3-mediated vascular defect. The growth-suppressive actions of β3, however, could be overcome by stimulation of pathways (Akt or vascular endothelial growth factor) commonly activated in GBM. These results show that tumor-specific αVβ3 overexpression has growth-suppressive effects in gliomas, but that these deleterious effects are mitigated by alterations common to αVβ3-overexpressing GBM.
The
Rgr
Oncogene Induces Tumorigenesis in Transgenic Mice
Authors: Jiménez María , de Castro Ignacio Pérez , Benet Marta , García Juan F. , Inghirami Giorgio , Pellicer Angel
Published: September 2005

To study the oncogenic potential of Rgr in vivo, we have generated several transgenic Rgr mouse lines, which express the oncogene under the control of different promoters. These studies revealed that Rgr expression leads to the generation of various pathological alterations, including fibrosarcomas, when its transgenic expression is restricted to nonlymphoid tissues. Moreover, the overall incidence and latency of fibrosarcomas were substantially increased and shortened, respectively, in a p15INK4b-defective background. More importantly, we also have demonstrated that Rgr expression in thymocytes of transgenic mice induces severe alterations in the development of the thymocytes, which eventually lead to a high incidence of thymic lymphomas. This study demonstrates that oncogenic Rgr can induce expression of p15INK4b and, more importantly, that both Rgr and p15INK4b cooperate in the malignant phenotype in vivo. These findings provide new insights into the tumorigenic role of Rgr as a potent oncogene and show that p15INK4b can act as a tumor suppressor gene.
Effect of Exercise on Serum Estrogens in Postmenopausal Women
Authors: McTiernan Anne , Tworoger Shelley S. , Ulrich Cornelia M. , Yasui Yutaka , Irwin Melinda L. , Rajan Kumar B. , Sorensen Bess , Rudolph Rebecca E. , Bowen Deborah , Stanczyk Frank Z. , Potter John D. , Schwartz Robert S.
Published: September 2005

Elevated circulating estrogens and a sedentary lifestyle increase risk for breast cancer. The effect of exercise on circulating estrogens in sedentary postmenopausal women is unknown. The objective of this study was to examine the effects of a 12-month moderate-intensity exercise intervention on serum estrogens. We randomly assigned 173 sedentary, overweight (body mass index > 24.0 kg/m2, body fat > 33%), postmenopausal women, ages 50–75 years, not using hormone therapy, living in the Seattle, Washington, area for the next year, and willing to be randomly assigned to an exercise intervention or stretching control group. The exercise intervention included facility and home-based exercise (45 min, 5 days/week moderate intensity sports/recreational exercise). A total of 170 (98.3%) women completed the study with exercisers averaging 171 min/week of exercise. After 3 months, exercisers experienced declines in estrone, estradiol, and free estradiol of 3.8, 7.7, and 8.2%, respectively, versus no change or increased concentrations in controls (P = 0.03, 0.07, and 0.02, respectively). At 12 months, the direction of effect remained the same, although the differences were no longer statistically significant. The effect was limited to women who lost body fat: women whose percentage of body fat [by dual energy x-ray absortiometry (DEXA)] decreased by ≥2% had statistically significant (comparing exercisers versus controls) decreases at 12 months of 11.9, 13.7, and 16.7% for serum estrone, estradiol, and free estradiol, respectively. We concluded that a 12-month moderate-intensity exercise intervention in postmenopausal women resulted in significant decreases in serum estrogens. The association between increased physical activity and reduced risk for postmenopausal breast cancer may be partly explained by effects on serum estrogens.
Increased Susceptibility of Poly(ADP-Ribose) Polymerase-1 Knockout Cells to Antitumor Triazoloacridone C-1305 Is Associated with Permanent G2 Cell Cycle Arrest
Authors: Węsierska-Gądek Józefa , Schloffer Daniela , Gueorguieva Marieta , Uhl Maria , Skladanowski Andrzej
Published: September 2005

Triazoloacridone C-1305 is a novel inhibitor of DNA topoisomerase II, which exhibits potent antitumor activity toward solid tumors. In this study, antiproliferative action of C-1305 and its close analog C-1533 was investigated in nontransformed mouse fibroblasts and two mutant cell lines in which the PARP-1 gene was specifically disrupted. Unexpectedly, C-1305 very strongly affected proliferation of cells lacking poly(ADP-ribose) polymerase-1 (PARP-1), whereas the action of less active compound C-1533 toward normal and PARP-1-negative cells was comparable. The IC50 concentration of C-1305 determined for PARP-1 knockout cells was ∼150-fold lower than that determined for cells with functional PARP-1. Both studied triazoloacridones exhibited very low direct cytotoxicity as evidenced by accumulation of 7-amino-actinomycin D, and only low levels of apoptosis were observed after a 24-h exposure to studied drugs. Analysis of DNA damage induced by C-1305 by the Comet assay showed that this drug induced very low levels of DNA strand breaks. C-1305 strongly affected cell cycle progression in normal and PARP-1 mutant cells and arrested both cell types in G2-M phase. However, the G2-M arrest induced by C-1305 was greatly prolonged in PARP-1-deficient cells as compared with normal fibroblasts. Together, these results show that mouse cells lacking PARP-1 are extremely sensitive to C-1305, a new topoisomerase II inhibitor. This is in striking contrast with previous reports in which PARP-1-deficient cells were shown to be resistant to classical topoisomerase II inhibitors. Our data also suggest that the PARP-1 status might be essential for the maintenance of the G2 arrest induced by C-1305.
Mutation of
hCDC4
Leads to Cell Cycle Deregulation of Cyclin E in Cancer
Authors: Reed Susanna Ekholm , Spruck Charles H. , Sangfelt Olle , van Drogen Frank , Mueller-Holzner Elisabeth , Widschwendter Martin , Zetterberg Anders , Reed Steven I.
Published: September 2005

hCDC4, the gene that encodes the F-box protein responsible for targeting cyclin E for ubiquitin-mediated proteolysis, has been found to be mutated in a number of primary cancers and cancer-derived cell lines. We have observed that functional inactivation of hCDC4 does not necessarily correlate with elevated levels of cyclin E in tumors. Here we show, however, that hCDC4 mutation in primary tumors correlates strongly with loss of cell cycle regulation of cyclin E. Similarly, a breast carcinoma-derived cell line mutated for hCDC4 exhibits cell cycle deregulation of cyclin E, but periodic expression is restored by reintroducing hCDC4 via retroviral transduction. Conversely, small interfering RNA-mediated silencing of hCdc4 deregulates cyclin E with respect to the cell cycle. These results indicate that hCdc4 function is an absolute prerequisite for cell cycle regulation of cyclin E levels, and loss of hCdc4 function is sufficient to deregulate cyclin E.
Laminin-Induced Signaling in Tumor Cells
Authors: Givant-Horwitz Vered , Davidson Ben , Reich Reuven
Published: September 2005

The expression of the Mr 67,000 laminin receptor, a nonintegrin laminin receptor, was found to be up-regulated in neoplastic cells and to directly correlate with invasion and metastatic potential. In the present study, we investigated the role of laminin receptor in mediating laminin effects and the involvement of the mitogen-activated protein kinases (MAPK) cascades and dual-specificity phosphatases in laminin signaling in human melanoma cells. Using stable transfection of A375SM melanoma cells, we established lines expressing reduced or elevated laminin receptor. The antisense-transfected cells demonstrated reduced attachment to laminin and reduced invasion through Matrigel-coated filters. In addition, both matrix metalloproteinase-2 (MMP-2) mRNA expression and activity were significantly reduced in the antisense-transfected cells. Antisense-transfected cells showed a reduction in mRNA level of the α6B integrin subunit isoform, whereas no change in the mRNA level of the α6A isoform was observed. We found that exogenous laminin reduced the phosphorylated (active) form of extracellular signal-regulated kinase, c-Jun NH2-terminal protein kinase, and p38 in all of the cells, irrespective of the expression of the laminin receptor. Furthermore, the phosphorylation of extracellular signal-regulated kinase, c-Jun NH2-terminal protein kinase, and p38 was significantly higher in the cell lines expressing reduced laminin receptor, regardless of the exposure to exogenous laminin. This increase of MAPK phosphorylation was accompanied by a significant reduction in MKP-1 phosphatase mRNA level and a significant increase in PAC-1 phosphatase mRNA level. In conclusion, our results confirm the involvement of the laminin receptor in different mechanisms related to tumor dissemination and provide first evidence of the involvement of MAPK and dual-specificity phosphatases in its signal transduction pathway.
Selectivity of an Oncolytic Herpes Simplex Virus for Cells Expressing the DF3/MUC1 Antigen
Authors: Kasuya Hideki , Pawlik Timothy M. , Mullen John T. , Donahue James M. , Nakamura Hideo , Chandrasekhar Soundararajalu , Kawasaki Hiroshi , Choi Enid , Tanabe Kenneth K.
Published: September 2005

Replication-conditional viruses destroy tumors in a process referred to as viral oncolysis. An important prerequisite for this cancer therapy strategy is use of viruses that replicate preferentially in neoplastic cells. In this study the DF3/MUC1 promoter/enhancer sequence is used to regulate expression of γ134.5 to drive replication of a Herpes simplex virus 1 (HSV-1) mutant (DF3γ34.5) preferentially in DF3/MUC1-positive cells. HSV-1 γ134.5 functions to dephosphorylate elongation initiation factor 2α, which is an important step for robust HSV-1 replication. After DF3γ34.5 infection of cells, elongation initiation factor 2α phosphatase activity and viral replication were observed preferentially in DF3/MUC1-positive cells but not in DF3/MUC1-negative cells. Regulation of γ134.5 function results in preferential replication in cancer cells that express DF3/MUC1, restricted biodistribution in vivo, and less toxicity as assessed by LD50. Preferential replication of DF3γ34.5 was observed in DF3/MUC1-positive liver tumors after intravascular perfusion of human liver specimens. DF3γ34.5 was effective against carcinoma xenografts in nude mice. Regulation of γ134.5 by the DF3/MUC1 promoter is a promising strategy for development of HSV-1 mutants for viral oncolysis.
V599EB-RAF is an Oncogene in Melanocytes
Authors: Wellbrock Claudia , Ogilvie Lesley , Hedley Douglas , Karasarides Maria , Martin Jan , Niculescu-Duvaz Dan , Springer Caroline J. , Marais Richard
Published: September 2005

The oncogenic version of B-RAF, V599EB-RAF, is found in approximately 70% of human melanomas. However, the role that this oncogene plays in melanoma is unclear because V559EB-RAF is also found in approximately 80% of benign nevi. We have examined the role of oncogenic B-RAF in the early stages of melanoma by expressing V599EB-RAF in cultured melanocytes. In these cells, V599EB-RAF induced constitutive mitogen activated ERK-activating kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, 12-O-tetradecanoylphorbol-13-acetate-independent growth, and tumorigenicity in nude mice. Intriguingly, in RAS-transformed melanocytes, B-RAF depletion did not block MEK-ERK signaling or cell cycle progression. Similarly, B-RAF depletion blocked MEK-ERK signaling in human melanoma cells harboring oncogenic B-RAF, but not in melanoma cells harboring oncogenic RAS. Thus, although B-RAF can act as a potent oncogene in the early stages of melanoma by signaling through MEK and ERK, it is not required for this signaling in RAS-transformed melanocytes due to innate redundancy within the pathway. These findings have important implications for future therapeutic strategies.