bluuebunny/crossref_metadata_2025 huggingface dataset:
Src Family Kinases Mediate Epidermal Growth Factor Receptor Ligand Cleavage, Proliferation, and Invasion of Head and Neck Cancer Cells
Authors: Zhang Qing , Thomas Sufi M. , Xi Sichuan , Smithgall Thomas E. , Siegfried Jill M. , Kamens Joanne , Gooding William E. , Grandis Jennifer Rubin
Published: September 2005

Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor α (TGF-α) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein–coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src–transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRP-induced proliferation or invasion of HNSCC cells. In addition to TGF-α release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-α and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-α and amphiregulin in HNSCC.
Autocrine Secretion of Fas Ligand Shields Tumor Cells from Fas-Mediated Killing by Cytotoxic Lymphocytes
Authors: Hallermalm Kristian , De Geer Anna , Kiessling Rolf , Levitsky Victor , Levitskaya Jelena
Published: September 2005

Mechanisms responsible for resistance of tumors to death receptor-mediated damage by cytotoxic lymphocytes are not well understood. Uveal melanoma cells expressed Fas but were insensitive to Fas triggering induced by bystander cytotoxic T lymphocytes or a Fas-specific agonistic antibody; this could not be ascribed to tumor counterattack against T cells or general resistance of the tumors to apoptosis. Treatment with inhibitors of metalloproteases rendered uveal melanomas sensitive to Fas-mediated cytotoxicity. Metalloprotease inhibitors did not affect the expression of Fas but increased the surface expression of Fas ligand (FasL), which correlated with the disappearance of soluble FasL from culture supernatants of tumor cells. FasL eluted from the surface of uveal melanomas specifically inhibited cytotoxic T lymphocyte lysis of tumor cells pretreated with an inhibitor of metalloproteases. In addition to uveal melanomas, a number of other tumor cell lines of various cellular origins were sensitized to Fas-mediated cytotoxicity by metalloprotease inhibitors. Our results show that autocrine secretion of FasL shields tumor cells from Fas-mediated killing by cytotoxic lymphocytes. This defines a novel mechanism of tumor escape from immune surveillance.
Dependence of the Cytotoxicity of DNA-Damaging Agents on the Mismatch Repair Status of Human Cells
Authors: Papouli Efterpi , Cejka Petr , Jiricny Josef
Published: September 2005

Mismatch repair (MMR) deficiency was reported to increase resistance of mammalian cells to killing by several genotoxic substances. However, although MMR-deficient cells are ∼100-fold more resistant to killing by SN1 type methylating agents than MMR-proficient controls, the sensitivity differences reported for the other agents were typically <2-fold. To test whether these differences were linked to factors other than MMR status, we studied the cytotoxicities of mitomycin C, chloroethylcyclohexyl nitrosourea, melphalan, psoralen-UVA, etoposide, camptothecin, ionizing radiation, and cis-dichlorodiaminoplatinum (cisplatin) in a strictly isogenic system. We now report that MMR deficiency reproducibly desensitized cells solely to cisplatin.
Identification of Human Autologous Cytotoxic T-Lymphocyte-Defined Osteosarcoma Gene That Encodes a Transcriptional Regulator, Papillomavirus Binding Factor
Authors: Tsukahara Tomohide , Nabeta Yuki , Kawaguchi Satoshi , Ikeda Hideyuki , Sato Yuriko , Shimozawa Kumiko , Ida Kazunori , Asanuma Hiroko , Hirohashi Yoshihiko , Torigoe Toshihiko , Hiraga Hiroaki , Nagoya Satoshi , Wada Takuro , Yamashita Toshihiko , Sato Noriyuki
Published: September 2005

The prognosis for patients with osteosarcoma who do not respond to current chemotherapy protocols still remains poor. Toward the goal of establishing efficacious peptide-based immunotherapy for those patients, we previously developed an autologous pair of CTLs and an osteosarcoma cell line. In the current study, we screened the cDNA library of this osteosarcoma cell line using an autologous CTL clone and identified cDNA encoding an antigen. The isolated cDNA was identical to papillomavirus binding factor (PBF), which was recently reported as a DNA binding transcription factor cooperating with RUNX1. Reverse transcription-PCR analysis revealed that PBF was expressed in 16 of 19 cases of bone and soft-tissue sarcoma cell lines (5 of 6 of osteosarcoma lines) and 57 of 76 sarcoma tissue samples (11 of 14 of osteosarcoma tissues). Also, PBF was expressed in 10 of 13 epithelial cancer cell lines and 20 of 34 of cancer tissues. In contrast, PBF was detected in some normal organs including ovary, pancreas, spleen, and liver by reverse transcription-PCR but was restricted in the cytoplasm by immunostaining and undetectable by Western blotting. Furthermore, a 12-mer peptide, CTACRWKKACQR, located at the COOH terminus of PBF, was found to be a minimum requirement for recognition by the CTL clone in the context of the HLA-B*5502 molecule. These findings suggest that PBF is a shared tumor-associated antigen, which may serve as a source of peptides applicable to peptide-based immunotherapy for osteosarcoma and other malignant tumors.
Dual-Agent Molecular Targeting of the Epidermal Growth Factor Receptor (EGFR)
Authors: Huang Shyhmin , Armstrong Eric A. , Benavente Sergio , Chinnaiyan Prakash , Harari Paul M.
Published: September 2005

Molecular inhibition of epidermal growth factor receptor (EGFR/HER1) signaling is under active investigation as a promising cancer treatment strategy. We examined the potency of EGFR inhibition achieved by combining anti-EGFR monoclonal antibody and tyrosine kinase inhibitor, which target extracellular and intracellular domains of the receptor, respectively. We specifically studied the combination of cetuximab (Erbitux, C225; ImClone Systems, New York, NY) with either gefitinib (Iressa, ZD1839; AstraZeneca, Macclesfield, UK) or erlotinib (Tarceva, OSI-774; Genentech, South San Francisco, CA) across a variety of human cancer cells. The combination of cetuximab plus gefitinib or erlotinib enhanced growth inhibition over that observed with either agent alone. As measured by immunostaining, inhibition of EGFR phosphorylation with the combination of cetuximab plus gefitinib or erlotinib was augmented over that obtained with single-agent therapy in head and neck (H&N) cancer cell lines. Phosphorylation inhibition of downstream effector molecules [mitogen-activated protein kinase (MAPK) and AKT] also was enhanced in tumor cells treated with the combination of cetuximab plus gefitinib or erlotinib. Flow cytometry and immunoblot analysis demonstrated that treatment of H&N tumor cells with cetuximab in combination with either gefitinib or erlotinib amplified the induction of apoptosis. Following establishment of cetuximab-resistant cell lines, we observed that gefitinib or erlotinib retained the capacity to inhibit growth of lung and H&N tumor cells that were highly resistant to cetuximab. Treatment with gefitinib or erlotinib, but not cetuximab, also could further inhibit the activation of downstream effectors of EGFR signaling in cetuximab-resistant cells, including MAPK and AKT. These data suggest that tyrosine kinase inhibitors may further modulate intracellular signaling that is not fully blocked by extracellular anti-EGFR antibody treatment. Finally, animal studies confirmed that single EGFR inhibitor treatment resulted in partial and transient tumor regression in human lung cancer xenografts. In contrast, more profound tumor regression and regrowth delay were observed in mice treated with the combination of cetuximab and gefitinib or erlotinib. Immunohistochemical staining, which demonstrated significant reduction of the proliferative marker proliferating cell nuclear antigen in mice treated with dual EGFR inhibitors, further supported this in vivo observation. Together, these data suggest that combined treatment with distinct EGFR inhibitory agents can augment the potency of EGFR signaling inhibition. This approach suggests potential new strategies to maximize effective target inhibition, which may improve the therapeutic ratio for anti-EGFR-targeted therapies in developing clinical trials.
A Comparative Analysis of Low-Dose Metronomic Cyclophosphamide Reveals Absent or Low-Grade Toxicity on Tissues Highly Sensitive to the Toxic Effects of Maximum Tolerated Dose Regimens
Authors: Emmenegger Urban , Man Shan , Shaked Yuval , Francia Giulio , Wong John W. , Hicklin Daniel J. , Kerbel Robert S.
Published: September 2005

The survival benefits of traditional maximum tolerated dose (MTD) cytotoxic therapy have been modest for the treatment of most types of metastatic malignancy and, moreover, often come with increased acute and chronic toxicity. Recent studies have demonstrated that the frequent administration of comparatively low doses of cytotoxic agents, with no extended breaks [low-dose metronomic (LDM) chemotherapy], may not only be at least as efficient as MTD therapy but also less toxic. This coincides with an apparent selectivity for “activated” endothelial cells of the tumor vasculature. However, the impact of LDM chemotherapy on the most sensitive target cell populations normally affected by MTD therapy (i.e., bone marrow progenitors, gut mucosa, and hair follicle cells) has not been analyzed in experimental detail. Therefore, we compared effects of LDM and MTD cyclophosphamide (CTX) on bone marrow and gut mucosa. Furthermore, we studied the potential impact of LDM CTX on angiogenesis in the context of wound healing and evidence of organ toxicity. We show absent or moderate hematologic and intestinal toxicity of LDM as opposed to MTD CTX. Of note was the finding of sustained lymphopenia, which is not unexpected given the use of CTX as immunosuppressive drug. There was no negative impact on wound healing or evidence of organ toxicity. LDM offers clear safety advantages over conventional MTD chemotherapy and therefore would appear to be ideal for long-term combination therapy with targeted antiangiogenic drugs.
Activation of Nuclear Factor κB
In vivo
Selectively Protects the Murine Small Intestine against Ionizing Radiation-Induced Damage
Authors: Wang Yong , Meng Aimin , Lang Hainan , Brown Stephen A. , Konopa Jennifer L. , Kindy Mark S. , Schmiedt Richard A. , Thompson John S. , Zhou Daohong
Published: September 2005

Exposure of mice to total body irradiation induces nuclear factor κB (NFκB) activation in a tissue-specific manner. In addition to the spleen, lymph nodes, and bone marrow, the tissues that exhibit NFκB activation now include the newly identified site of the intestinal epithelial cells. NFκB activated by total body irradiation mainly consists of NFκB p50/RelA heterodimers, and genetically targeted disruption of the NFκB p50 gene in mice significantly decreased the activation. By comparing tissue damage and lethality in wild-type and NFκB p50 knockout (p50−/−) mice after they were exposed to increasing doses of total body irradiation, we additionally examined the role of NFκB activation in total body irradiation-induced tissue damage. The results show that p50−/− mice are more sensitive to total body irradiation-induced lethality than wild-type mice (LD50/Day 7: wild-type = 13.12 Gy versus p50−/− = 7.75 Gy and LD50/Day 30: wild-type = 9.31 Gy versus p50−/− = 7.81 Gy). The increased radiosensitivity of p50−/− mice was associated with an elevated level of apoptosis in intestinal epithelial cells and decreased survival of the small intestinal crypts compared with wild-type mice (P < 0.01). In addition, RelA/TNFR1-deficient (RelA/TNFR1−/−) mice also exhibited a significant increase in intestinal epithelial cell apoptosis after they were exposed to total body irradiation as compared with TNFR1-deficient (TNFR1−/−) mice (P < 0.01). In contrast, no significant increase in total body irradiation-induced apoptosis or tissue injury was observed in bone marrow cells, spleen lymphocytes, and the liver, heart, lung, and kidney of p50−/− mice in comparison with wild-type mice. These findings indicate that activation of NFκB selectively protects the small intestine against ionizing radiation-induced damage.
The Gap Junction Protein Connexin32 Is a Mouse Lung Tumor Suppressor
Authors: King Timothy J. , Lampe Paul D.
Published: September 2005

Although loss of connexin expression and/or gap junction intercellular communication correlates with decreased growth control and increased neoplastic potential, there is limited evidence directly linking gap junction intercellular communication function with tumor suppression in situ. Here, we show for the first time that a gap junction protein, connexin32 (Cx32), acts as a lung tumor suppressor in a mouse model. Cx32-deficient nontumorous lung tissue exhibited an increased proliferative index (P < 0.001), and, after exposure to the carcinogen diethylnitrosamine, Cx32-deficient mice exhibited a highly statistically significant (P < 0.001) increase in bronchioloalveolar lung tumor incidence (28 of 45, 62%) and a 45% increase in average multiplicity compared with wild-type mice (7 of 29, 24%). Tumors from Cx32-deficient mice also showed increased activation of mitogen-activated protein kinase (P < 0.001) compared with wild-type tumors, implicating this signaling pathway in Cx32/gap junction intercellular communication-associated lung tumorigenesis.
Dendritic Cells Pulsed with an Anti-Idiotype Antibody Mimicking Carcinoembryonic Antigen (CEA) Can Reverse Immunological Tolerance to CEA and Induce Antitumor Immunity in CEA Transgenic Mice
Authors: Saha Asim , Chatterjee Sunil K. , Foon Kenneth A. , Primus F. James , Sreedharan Sunil , Mohanty Kartik , Bhattacharya-Chatterjee Malaya
Published: September 2005

In this report, we have studied the immunogenicity of the nominal antigen, carcinoembryonic antigen (CEA), and that of an anti-idiotype antibody, 3H1, which mimics CEA and can be used as a surrogate for CEA. We have demonstrated that immunization of CEA transgenic mice with bone marrow-derived mature dendritic cells (DC) loaded with anti-idiotype 3H1 or CEA could reverse CEA unresponsiveness and result in the induction of CEA-specific immune responses and the rejection of CEA-transfected MC-38 colon carcinoma cells, C15. Immunized mice splenocytes proliferated in an antigen-specific manner by a mechanism dependent on the functions of CD4, MHC II, B7–2, CD40, CD28, and CD25. However, immune splenic lymphocytes isolated from 3H1-DC-vaccinated mice when stimulated in vitro with 3H1 or CEA secreted significantly higher levels of Th1 cytokines than did CEA-DC vaccinated mice. DC vaccination also induced antigen-specific effector CD8+ T cells capable of expressing interluekin-2, IFN-γ, and tumor necrosis factor (TNF)-α and displayed cytotoxic activity against C15 cells in an MHC class I-restricted manner. 3H1-DC vaccination resulted in augmented CTL responses and the elevated expression of CD69, CD25, and CD28 on CD8+ CTLs. The immune responses developed in 3H1-DC-immunized mice resulted in rejection of C15 tumor cells in nearly 100% of experimental mice, whereas only 40% of experimental mice immunized with CEA-DC were protected from C15 tumor growth. These findings suggest that under the experimental conditions used, 3H1-DC vaccination was better than CEA-DC vaccination in breaking immune tolerance to CEA and inducing protective antitumor immune responses in this murine model transgenic for human CEA.
CC Chemokine Ligand 25 Enhances Resistance to Apoptosis in CD4+ T Cells from Patients with T-Cell Lineage Acute and Chronic Lymphocytic Leukemia by Means of Livin Activation
Authors: Qiuping Zhang , Jei Xiong , Youxin Jin , Wei Ju , Chun Liu , Jin Wang , Qun Wu , Yan Liu , Chunsong Hu , Mingzhen Yang , Qingping Gao , Kejian Zhang , Zhimin Sun , Qun Li , Junyan Liu , Jinquan Tan
Published: September 2005

We investigated CD4 and CD8 double-positive thymocytes, CD4+ T cells from typical patients with T-cell lineage acute lymphocytic leukemia (T-ALL) and T cell lineage chronic lymphocytic leukemia (T-CLL), and MOLT4 T cells in terms of CC chemokine ligand 25 (CCL25) functions of induction of resistance to tumor necrosis factor α (TNF-α)–mediated apoptosis. We found that CCL25 selectively enhanced resistance to TNF-α–mediated apoptosis in T-ALL and T-CLL CD4+ T cells as well as in MOLT4 T cells, but CD4 and CD8 double-positive thymocytes did not. One member protein of the inhibitor of apoptosis protein (IAP) family, Livin, was selectively expressed in the malignant cells at higher levels, particularly in T-ALL CD4+ T cells, in comparison with the expression in CD4 and CD8 double-positive thymocytes. After stimulation with CCL25 and apoptotic induction with TNF-α, the expression levels of Livin in these malignant cells were significantly increased. CCL25/thymus-expressed chemokine (TECK), by means of CC chemokine receptor 9 (CCR9) ligation, selectively activated Livin to enhance resistance to TNF-α–mediated apoptosis in c-jun-NH2-kinase 1 (JNK1) kinase-dependent manner. These findings suggested differential functions of CCR9/CCL25 in distinct types of cells. CD4 and CD8 double-positive thymocytes used CCR9/CCL25 for migration, homing, development, maturation, selection, cell homeostasis, whereas malignant cells, particularly T-ALL CD4+ T cells, used CCR9/CCL25 for infiltration, resistance to apoptosis, and inappropriate proliferation.
Acetylcholinesterase Plays a Pivotal Role in Apoptosome Formation
Authors: Park Sang Eun , Kim Nam Deuk , Yoo Young Hyun
Published: September 2005

Although a recent study (Zhang et al. Cell Death Differ 2002; 9; 790–800) presented that acetylcholinesterase (AChE) might be an important common component in leading to various types of apoptosis, the molecular mechanism, by which AChE functions, had remained elusive before that study. We explored the role of AChE in apoptosis by silencing the AChE gene. Silencing of the AChE gene abolished the expression of AChE and prevented caspase-9 activation, decrease of cell viability, nuclear condensation and poly(adenosine diphosphate-ribose) polymerase cleavage but not mitochondrial events. Importantly, silencing of the AChE gene blocked the interaction between apoptotic protease-activating factor-1 and cytochrome c. Here we propose that AChE plays a pivotal role in the formation of apoptosome.
Functional
STK15
Phe31Ile Polymorphism Is Associated with the Occurrence and Advanced Disease Status of Esophageal Squamous Cell Carcinoma
Authors: Miao Xiaoping , Sun Tong , Wang Yonggang , Zhang Xuemei , Tan Wen , Lin Dongxin
Published: September 2005

STK15/BTAK/Aurora-A involved in regulating centrosomes and chromosome segregation is amplified and overexpressed in human cancers. A T91A polymorphism in STK15 causes Phe31Ile substitution, and the 31Ile variant has been shown to be preferentially amplified and associated with degree of aneuploidy in human tumors. We genotyped 656 patients with esophageal squamous cell carcinoma (ESCC) and 656 controls for the polymorphism to examine the hypothesis that the STK15 variation may affect individual susceptibility to the occurrence and aggression of ESCC. It was found that the Ile/Ile genotype was significantly associated with increased risk of ESCC occurrence [odds ratio (OR) = 1.97, 95% confidence interval (CI) = 1.36–2.85] compared with the Phe/Phe genotype. The 31Ile allele frequency significantly increased as ESCC stage increased (trend test, P = 0.006). Patients with the Ile/Ile genotype had an increased risk for invasive disease (stage II-IV; OR = 2.13, 95% CI = 1.04–4.39) or metastatic disease (stage III and IV; OR = 2.31, 95% CI = 1.06–5.05) compared with those with the Phe/Phe genotype. A positive correlation between the Ile/Ile genotype and high ESCC grade was also observed. Our results demonstrate for the first time that the STK15 polymorphism is a genetic susceptibility factor for the occurrence and aggression of ESCC.
Kaposi’s Sarcoma-Associated Herpesvirus Induction of Chromosome Instability in Primary Human Endothelial Cells
Authors: Pan Hongyi , Zhou Fuchun , Gao Shou-Jiang
Published: September 2005

Chromosome instability contributes to the multistep oncogenesis of cancer cells. Kaposi’s sarcoma (KS), an angiogenic vascular spindle cancer of endothelial cells, displays stage advancement with lesions at early stage being hyperproliferative, whereas lesions at late stage are clonal or multiclonal and can exhibit a neoplastic nature and chromosome instability. Although infection with KS-associated herpesvirus (KSHV) has been associated with the initiation and promotion of KS, the mechanism of KS neoplastic transformation remains unclear. We show that KSHV infection of primary human umbilical vein endothelial cells induces abnormal mitotic spindles and centrosome duplication. As a result, KSHV-infected cells manifest chromosome instability, including chromosomal misalignments and laggings, mitotic bridges, and formation of micronuclei and multinucleation. Our results indicate that KSHV infection could predispose cells to malignant transformation through induction of genomic instability and contributes to the development of KS.
Nitric Oxide Confers Therapeutic Activity to Dendritic Cells in a Mouse Model of Melanoma
Authors: Perrotta Cristiana , Falcone Sestina , Capobianco Annalisa , Camporeale Annalisa , Sciorati Clara , Palma Clara De , Pisconti Addolorata , Rovere-Querini Patrizia , Bellone Matteo , Manfredi Angelo A. , Clementi Emilio
Published: September 2005

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.
Augmenting Chemosensitivity of Malignant Melanoma Tumors via Proteasome Inhibition
Authors: Amiri Katayoun I. , Horton Linda W. , LaFleur Bonnie J. , Sosman Jeffrey A. , Richmond Ann
Published: September 2005

Melanoma poses a great challenge to patients, oncologists, and biologists because of its nearly universal resistance to chemotherapy. Many studies have shown that nuclear factor κB is constitutively activated in melanoma, thereby promoting the proliferation of melanoma cells by inhibiting the apoptotic responses to chemotherapy. Nuclear factor κB activity is regulated by phosphorylation and subsequent degradation of inhibitor of nuclear factor κB by the ubiquitin-proteasome pathway. In this study, we show that the novel proteasome inhibitor, bortezomib, inhibited the growth of melanoma cells in vitro at a concentration range of 0.1–10 nm and in combination with the chemotherapeutic agent temozolomide, the inhibitory effect on melanoma cell growth was even more prominent. Data from a murine model showed reduced tumor growth when bortezomib was administered to human melanoma tumors. Strikingly, animals receiving bortezomib in combination with temozolomide achieved complete remission of palpable tumors after only 30 days of therapy, lasting >200 days. Our data indicate strongly that bortezomib in combination with chemotherapeutic agents should be studied additionally for the treatment of melanoma.
Synergistic Suppression of Microtubule Dynamics by Discodermolide and Paclitaxel in Non-Small Cell Lung Carcinoma Cells
Authors: Honore Stéphane , Kamath Kathy , Braguer Diane , Horwitz Susan Band , Wilson Leslie , Briand Claudette , Jordan Mary Ann
Published: September 2005

Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978–1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC50s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G2-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.
Epidermal Growth Factor Receptor Regulates Aberrant Expression of Insulin-Like Growth Factor-Binding Protein 3
Authors: Takaoka Munenori , Harada Hideki , Andl Claudia D. , Oyama Kenji , Naomoto Yoshio , Dempsey Kelly L. , Klein-Szanto Andres J. , El-Deiry Wafik S. , Grimberg Adda , Nakagawa Hiroshi
Published: September 2005

Epidermal growth factor receptor (EGFR) is frequently overexpressed in esophageal carcinoma and its precursor lesions. To gain insights into how EGFR overexpression affects cellular functions in primary human esophageal cells, we performed gene expression profiling and identified insulin-like growth factor-binding protein (IGFBP)-3 as the most up-regulated gene. IGFBP-3 regulates cell proliferation through both insulin-like growth factor-dependent and independent mechanisms. We found that IGFBP-3 mRNA and protein expression was increased in EGFR-overexpressing primary and immortalized human esophageal cells. IGFBP-3 was also up-regulated in EGFR-overexpressing cells in organotypic culture and in EGFR transgenic mice. Furthermore, IGFBP-3 mRNA was overexpressed in 80% of primary esophageal squamous cell carcinomas and 60% of primary esophageal adenocarcinomas. Concomitant up-regulation of EGFR and IGFBP-3 was observed in 60% of primary esophageal squamous cell carcinomas. Immunohistochemistry revealed cytoplasmic localization of IGFBP-3 in the preponderance of preneoplastic and neoplastic esophageal lesions. IGFBP-3 was also overexpressed in esophageal cancer cell lines at both mRNA (60%) and protein (40%) levels. IGFBP-3 secreted by cancer cells was capable of binding to insulin-like growth factor I. Functionally, epidermal growth factor appeared to regulate IGFBP-3 expression in esophageal cancer cell lines. Finally, suppression of IGFBP-3 by small interfering RNA augmented cell proliferation, suggesting that IGFBP-3 may inhibit tumor cell proliferation as a negative feedback mechanism. In aggregate, we have identified for the first time that IGFBP-3 is an aberrantly regulated gene through the EGFR signaling pathway and it may modulate EGFR effects during carcinogenesis.
Functional Correlation of Trophinin Expression with the Malignancy of Testicular Germ Cell Tumor
Authors: Hatakeyama Shingo , Ohyama Chikara , Minagawa Shingo , Inoue Takamitsu , Kakinuma Hideaki , Kyan Atsushi , Arai Yoichi , Suga Tomoaki , Nakayama Jun , Kato Tetsuro , Habuchi Tomonori , Fukuda Michiko N.
Published: September 2005

Trophinin is a membrane protein that is potentially involved in human embryo implantation by mediating homophillic cell adhesion between trophoblastic cells and endometrial cells. Trophinin expression by maternal cells may be induced by the embryo that secretes human chorionic gonadotropin (hCG). Because the process of tumor metastasis resembles that of trophoblast invasion and proliferation during embryo implantation, we hypothesized that testicular cancers that synthesize hCG express trophinin thus becoming aggressive trophoblast-like cells. We screened paraffin-embedded orchiectomy specimens of 158 patients with testicular germ cell tumor by immunohistochemistry using antitrophinin antibody. This screening identified trophinin-positive specimens with the frequencies 39 of 91 (43%) in stage I, 14 of 24 (58%) in stage II, and 41 of 43 (95%) in stage III (P < 0.001). Thus, trophinin expression positively correlates with clinical stage. Remarkably, trophinin was found in all of the cases (33 of 33) with lung metastasis. The levels of serum hCG-β were significantly higher in the patients with trophinin-positive tumors than those with trophinin-negative tumors (P = 0.004). To determine whether trophinin promotes aggressiveness of the cell, trophinin-negative human seminona cell line JKT-1 was stably transfected with a mammalian expression vector containing trophinin cDNA. In vitro assays revealed that trophinin-expressing JKT-1-Tro cells are more invasive than JKT-1-mock cells, whereas there are no differences between JKT-1-Tro and JKT-1-mock in their proliferation activity. Upon orthotopic inoculation to athymic nude mice, JKT-1-Tro cells exhibited i.p. metastases in all of the mice (n = 5), whereas JKT-1-mock produced no metastases (n = 5). These results suggest strongly that trophinin enhances invasiveness of the cells and promotes metastasis of testicular germ cell tumor.
Highly Successful Therapeutic Vaccinations Combining Dendritic Cells and Tumor Cells Secreting Granulocyte Macrophage Colony-stimulating Factor
Authors: Driessens Gregory , Hamdane Malika , Cool Vincent , Velu Thierry , Bruyns Catherine
Published: September 2005

In an attempt to induce potent immune antitumor activities, we investigated, within the rat 9L gliosarcoma model, distal therapeutic vaccinations associating three therapies: dendritic cell vaccination, intratumoral granulocyte macrophage colony-stimulating factor (GM-CSF) gene transfer, and tumor apoptosis induction. Vaccines of dendritic cells coinjected with processed GM-CSF secreting 9L cells induced systemic responses, resulting in the complete regression of distant preimplanted 9L tumor masses in, with the best strategy, 94% of male rats. All of the cured rats developed a long-term resistance to a rechallenge with parental cells. The curative responses were correlated with the detection of elevated specific cytotoxic activities and a CD4+, CD8+ T cell-, and natural killer (NK) cell-mediated IFN-γ production. The survival rate of the rat seemed more directly linked to the amount of GM-CSF secreted by the transduced tumor cells, which in turn depended on the toxicity of the apoptosis-inducing treatment, than to the level of apoptosis induced. Unexpectedly, alive GM-CSF secreting 9L cells became apoptotic when injected in vivo. Thus we documented the positive role of apoptosis in the induction of therapeutic antitumor responses by comparing, at equal GM-CSF exogenous supply, the effects of dendritic cells coinjected with apoptotic or necrotic 9L cells. The data showed the superior therapeutic efficiency of combined vaccines containing apoptotic tumor cells. In conclusion, vaccinations with dendritic cells associated with apoptotic tumor cells secreting GM-CSF show a very high therapeutic potency that should show promise for the treatment of human cancer.
P-Cadherin Is Up-Regulated by the Antiestrogen ICI 182,780 and Promotes Invasion of Human Breast Cancer Cells
Authors: Paredes Joana , Stove Christophe , Stove Veronique , Milanezi Fernanda , Van Marck Veerle , Derycke Lara , Mareel Marc , Bracke Marc , Schmitt Fernando
Published: September 2005

P-cadherin expression in breast carcinomas has been associated with tumors of high histologic grade and lacking estrogen receptor-α, suggesting a link between these proteins. In the MCF-7/AZ breast cancer cell line, blocking estrogen receptor-α signaling with the antiestrogen ICI 182,780 induced an increase of P-cadherin, which coincided with induction of in vitro invasion. Retroviral transduction of MCF-7/AZ cells, as well as HEK 293T cells, showed the proinvasive activity of P-cadherin, which requires the juxtamembrane domain of its cytoplasmic tail. This study establishes a direct link between P-cadherin expression and the lack of estrogen receptor-α signaling in breast cancer cells and suggests a role for P-cadherin in invasion, through its interaction with proteins bound to the juxtamembrane domain.
Gli Proteins Up-Regulate the Expression of Basonuclin in Basal Cell Carcinoma
Authors: Cui Chunhua , Elsam Thomas , Tian Qinjie , Seykora John T. , Grachtchouk Marina , Dlugosz Andrzej , Tseng Hung
Published: September 2005

Tumorigenesis is frequently accompanied by enhanced rRNA transcription, but the signaling mechanisms responsible for such enhancement remain unclear. Here, we report evidence suggesting a novel link between deregulated Hedgehog signaling and the augmented rRNA transcription in cancer. Aberrant activation of the Hedgehog pathway in keratinocytes is a hallmark of basal cell carcinoma (BCC), the most common cancer in light-skinned individuals. We show that Gli proteins, downstream effectors of the Hedgehog pathway, increase expression of a novel rRNA gene (rDNA) transcription factor, basonuclin, whose expression is markedly elevated in BCCs. The promoter of the human basonuclin gene contains a Gli-binding site, which is required for Gli protein binding and transcriptional activation. We show also that the level of 47S pre-rRNA is much higher in BCCs than in normal epidermis, suggesting an accelerated rRNA transcription in the neoplastic cells. Within BCC, those cells expressing the highest level of basonuclin also exhibit the greatest increase in 47S pre-rRNA, consistent with a role for basonuclin in increasing rRNA transcription in these cells. Our data suggest that Hedgehog-Gli pathway enhances rRNA transcription in BCC by increasing basonuclin gene expression.
Oncogenic K-ras Drives Cell Cycle Progression and Phenotypic Conversion of Primary Pancreatic Duct Epithelial Cells
Authors: Agbunag Cristina , Bar-Sagi Dafna
Published: September 2005

We have established a primary pancreatic duct epithelial cell culture (PDEC) system to investigate the relationship between oncogenic activation of K-ras and pancreatic ductal tumorigenesis. We have found that the acute introduction of physiological levels of oncogenic K-ras (K-rasV12) into quiescent PDECs stimulates S-phase entry and induces a pronounced increase in cell size. Both effects are dependent on the functional integrity of the phosphatidylinositol 3′-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. In addition, K-rasV12 promotes the loss of epithelial E-cadherin and the gain of mesenchymal N-cadherin in PDEC. Our observations indicate that the oncogenic activation of K-ras is sufficient to elicit mitogenic and morphogenic responses in pancreatic ductal cells and hence is likely to play an instructive role in the initiation of pancreatic ductal adenocarcinoma.
Profiling the Evolution of Human Metastatic Bladder Cancer
Authors: Nicholson Brian E. , Frierson Henry F. , Conaway Mark R. , Seraj Jabed M. , Harding Michael A. , Hampton Garret M. , Theodorescu Dan
Published: September 2005

Pulmonary metastases frequently develop in patients with aggressive bladder cancer, yet investigation of this process at the molecular level suffers from the poor availability of human metastatic tumor tissue and the absence of suitable animal models. To address this, we developed progressively more metastatic human bladder cancer cell lines and an in vivo bladder-cancer lung-metastasis model, and we successfully used these to identify genes of which the expression levels change according to the degree of pulmonary metastatic potential. By initially intravenously injecting the poorly metastatic T24T human urothelial cancer cells into nude mice, and then serially reintroducing and reisolating the human tumor cells from the resultant mouse lung tumors, three derivative human lines with increasingly metastatic phenotypes, designated FL1, FL2, and FL3, were sequentially isolated. To identify the genes associated with the most lung-metastatic phenotype, the RNA complement from the parental and derivative cells was evaluated with oligonucleotide microarrays. In doing so, we found 121 genes to be progressively up-regulated during the transition from T24T to FL3, whereas 43 genes were progressively down-regulated. As expected, many of the genes identified in these groups could, according to the ascribed functions of their protein product, theoretically participate in tissue invasion and metastasis. In addition, the magnitude of gene expression changes observed during the metastatic transition correlated with the in vivo propensity for earlier lung colonization and decreased host survival. To additionally define which genes found in the experimental system were of relevance to human bladder cancer lung metastasis, we evaluated gene expression profiles of 23 primary human bladder tumors of various stages and grades, and then we compared these gene expression profiles to the altered profiles in our model cell lines. Here we found that the expression of epiregulin, urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)14, and tissue inhibitor of metalloproteinase (TIMP-2) were consistently and progressively up-regulated when viewed as a function of tumor stage in tissues of patients versus the metastatic potential seen in the mouse lung model. The strong correlation of these four markers between the experimental and clinical situations helps validate this system as a useful tool for the study of lung metastasis and defines targets of therapy that may reduce the incidence of this process in patients.
Growth and Survival Mechanisms Associated with Perineural Invasion in Prostate Cancer
Authors: Ayala Gustavo E. , Dai Hong , Ittmann Michael , Li Rile , Powell Michael , Frolov Anna , Wheeler Thomas M. , Thompson Timothy C. , Rowley David
Published: September 2005

Perineural invasion (PNI) is the major mechanism of prostate cancer spread outside the prostate. Apoptotic and proliferation indices were determined in PNI cells using the PNI in vitro model and human PNI in tissue microarrays. RNA was extracted from the PNI model and controls and evaluated by cDNA microarray analysis. Differential expression of candidate genes was confirmed by real-time quantitative PCR, fluorescence, and immunohistochemistry using tissue microarrays. Genistein and BAY 11-7085 were added to the supernatant of cocultures and controls in microchamber cultures. The significance of nuclear factor κB (NFκB) nuclear translocation in human PNI was analyzed using Kaplan-Meier analysis. An increase in proliferation and a decrease in apoptosis were observed in human PNI cells and the PNI model as compared with controls. Three of 15 genes up-regulated in the cDNA microarray were involved in the apoptosis signaling pathway (NFκB), and its downstream targets defender against cell death 1 and PIM-2. The increase was corroborated by real-time quantitative PCR and immunofluorescence. NFκB nuclear translocation was seen in the in vitro model and human tissues, where strong nuclear expression was associated with a decrease in recurrence-free survival. Addition of genistein and BAY 11-7085 resulted in a decrease in NFκB, PIM-2 and defender against cell death 1 as well as a reversal of the inhibition of apoptosis. This is the first description of a biological mechanism and functional significance of PNI. Cancer cells in a perineural location acquire a survival and growth advantage using a NFκB survival pathway. Targeting PNI might help detain local spread of the tumor and influence survival.
TEL2, an ETS Factor Expressed in Human Leukemia, Regulates Monocytic Differentiation of U937 Cells and Blocks the Inhibitory Effect of TEL1 on Ras-Induced Cellular Transformation
Authors: Kawagoe Hiroyuki , Potter Mark , Ellis John , Grosveld Gerard C.
Published: September 2005

TEL2 is a member of the ETS family of transcription factors, which is highly similar to TEL1/ETV6. It binds to DNA via the ETS domain and interacts with itself or TEL1 via the pointed domain. The expression of TEL2 in normal and leukemic hematopoietic cells suggests a role in hematopoietic development. In this article, we describe the role of TEL2 in hematopoietic differentiation and cellular transformation. Quantitative reverse transcription-PCR showed that the expression of TEL2 mRNA was down-regulated during monocytic differentiation of U937 and HL60 induced by 1,25-(OH)2 vitamin D3 and 12-O-tetradecanoylphorbol 13-acetate, respectively. Overexpression of TEL2 in U937 cells inhibited differentiation induced by vitamin D3. In contrast, overexpression of a TEL2 mutant lacking either the pointed domain or a functional ETS domain induced both differentiation of U937 cells and inhibited their growth in vitro and in vivo. In addition, these mutants blocked TEL2-mediated transcriptional repression of a synthetic promoter containing TEL2 binding sites. These data suggest that dominant-negative inhibition of TEL2 might cause differentiation. Quantitative reverse transcription-PCR demonstrated that TEL2 is expressed at higher level in some primary human leukemia samples than in normal bone marrow. Furthermore, overexpression of TEL2 in NIH3T3-UCLA cells blocked the inhibitory effect of TEL1 on Ras-induced cellular transformation. These results suggest that TEL2 may play an important role in hematopoiesis and oncogenesis.
Potentiation of Cytotoxicity of Topoisomerase I Poison by Concurrent and Sequential Treatment with the Checkpoint Inhibitor UCN-01 Involves Disparate Mechanisms Resulting in Either p53-Independent Clonogenic Suppression or p53-Dependent Mitotic Catastrophe
Authors: Tse Archie N. , Schwartz Gary K.
Published: September 2005

UCN-01 is a potent inhibitor of the S- and G2-M-phase cell cycle checkpoints by targeting chk1 and possibly chk2 kinases. It has been shown in some, but not all, instances that UCN-01 potentiates the cytotoxicity of DNA-damaging agents selectively in p53-defective cells. We have investigated this concept in HCT116 colon cancer cells treated with the topoisomerase I poison SN-38. SN-38 alone induced a senescence-like sustained G2 arrest without apoptosis. Sequential treatment with SN-38 followed by UCN-01 resulted in enhancement of cytotoxicity by apoptosis assay, whereas the reverse sequence or concurrent treatment did not potentiate apoptosis. Real-time visualization of HCT116 cells labeled with green fluorescent protein-histone 2B or green fluorescent protein-α-tubulin revealed that sequential treatment resulted in G2 checkpoint abrogation, and cells entered an aberrant mitosis despite normal assembly of bipolar spindles, resulting in either apoptosis or formation of micronucleated cells. Although p53-null cells were clearly more sensitive than parental HCT116 to undergoing checkpoint abrogation and mitotic death after sequential treatment, this was not accompanied by an increased inhibition of clonogenicity over that induced by SN-38 alone. Conversely, concurrent treatment with SN-38 and UCN-01 resulted in S-phase checkpoint override, an amplified DNA damage response including increased phosphorylation of the DNA double-strand breakage marker H2AX and augmentation of clonogenic inhibition, which was independent of p53. Thus, reported discrepancies in the pharmacology of UCN-01 and the influence of p53 status on treatment outcome appears to stem, in part, from the different schedules used, the specific checkpoints examined, and the assays used to assess cytotoxicity. Moreover, checkpoint abrogation and subsequent apoptosis induced by UCN-01 do not necessarily correlate with reproductive cell death.
Molecular Imaging of Temporal Dynamics and Spatial Heterogeneity of Hypoxia-Inducible Factor-1 Signal Transduction Activity in Tumors in Living Mice
Authors: Serganova Inna , Doubrovin Michael , Vider Jelena , Ponomarev Vladimir , Soghomonyan Suren , Beresten Tatiana , Ageyeva Ludmila , Serganov Alexander , Cai Shangde , Balatoni Julius , Blasberg Ronald , Gelovani Juri
Published: September 2005

Tumor hypoxia is a spatially and temporally heterogeneous phenomenon, which results from several tumor and host tissue-specific processes. To study the dynamics and spatial heterogeneity of hypoxia-inducible factor-1 (HIF-1)-specific transcriptional activity in tumors, we used repetitive noninvasive positron emission tomography (PET) imaging of hypoxia-induced HIF-1 transcriptional activity in tumors in living mice. This approach uses a novel retroviral vector bearing a HIF-1–inducible “sensor” reporter gene (HSV1-tk/GFP fusion) and a constitutively expressed “beacon” reporter gene (DsRed2/XPRT). C6 glioma cells transduced with this multireporter system revealed dose-dependent patterns in temporal dynamics of HIF-1 transcriptional activity induced by either CoCl2 or decreased atmospheric oxygen concentration. Multicellular spheroids of C6 reporter cells developed a hypoxic core when >350 μm in diameter. 18F-2′-fluoro-2′deoxy-1β-D-arabionofuranosyl-5-ethyl-uracil (FEAU) PET revealed spatial heterogeneity of HIF-1 transcriptional activity in reporter xenografts in mice as a function of size or ischemia-reperfusion injury. With increasing tumor diameter (>3 mm), a marked increase in HIF-1 transcriptional activity was observed in the core regions of tumors. Even a moderate ischemia-reperfusion injury in small C6 tumors caused a rapid induction of HIF-1 transcriptional activity, which persisted for a long time because of the inability of C6 tumors to rapidly compensate acute changes in tumor microcirculation.