Primary Tumor Cell Cultures Research Articles

Tamoxifen-induced ER-α–SRC-3 interaction in HER2 positive human breast cancer; a possible mechanism for ER isoform specific recurrence

Differential signalling between the two oestrogen receptor (ER) isoforms in the presence of tamoxifen has been described. We hypothesise that differential recruitment of the steroid receptor co-activator, SRC-3 to ER-alpha and ER-beta may in part explain associations between ER isoforms and response to endocrine treatment. SRC-3 was localised within epithelial cells of breast tumour tissue and was co-localised with ER-alpha and ER-beta, (n=112). Expression of SRC-3 was found to be positively associated with ER-alpha (P=0.0021) and inversely with ER-beta (P<0.0001). Uniquely, this study utilises primary cell cultures derived from patient tumours, thus providing samples not readily available in most molecular model systems. These samples have enabled us to investigate the influence of growth factor pathways on steroid receptor-co-activator interactions. In HER2 (human epidermal growth factor receptor 2) positive primary tumour cell cultures 17beta-estradiol induced a decrease in SRC-3, whereas upregulated SRC-3 expression. Furthermore, treatment with tamoxifen-induced SRC-3 recruitment to the ER-oestrogen response element and enhanced interaction between SRC-3 and ER-alpha, but not ER-beta. Knockdown of SRC-3 results in a concomitant loss of expression of the oestrogen target gene pS2. Furthermore, silencing of SRC-3 resensitizes endocrine resistant, HER2 positive cells to the anti-proliferative effects of tamoxifen. The ability of ER-alpha, but not ER-beta to recruit SRC-3 in the presence of tamoxifen may in part explain the differential ER isoform associations with recurrence in human breast cancer.

Fibronectin is distinctly downregulated in murine mammary adenocarcinoma cells with high metastatic potential

In the present work we used a murine mammary cancer model of two related adenocarcinomas with different lung metastasizing abilities, to compare their global gene expression profiles. Clontech Atlas mouse cDNA microarrays of primary cultured tumor cells were employed to identify genes that are modulated in the more metastatic variant MM3 relative to its parental tumor M3. A total of 88 from 1,176 genes were differentially expressed in MM3 primary cultures, most of them (n=86) were upregulated. Genes were grouped according to their functions as associated with signal transduction and transcription regulation (e.g. Stat1 and Zfp 92), with cell adhesion and motility (cadherin 1, fibronectin), with invasion and angiogenesis (uPA, 72 kDa MMP2), with the regulation of cell proliferation and cell death (cyclins G and A2, TNF), and also included growth factors and receptors, oncogenes and tumor suppressors genes (p107, TGFbeta2, TBR-I, PDGFR). Only 2 genes, TTF1 and fibronectin (FN), showed a significant downregulation. Notably FN expression, loss of which has been associated with a malignant phenotype, was reduced about 19-fold in the more metastatic MM3 cells. Previously known differences in expression patterns associated with the metastatic capacity of MM3 and M3 adenocarcinomas, including downregulation of FN or upregulated expression of TGFbeta and proteases, were confirmed by the array data. The fact that FN was one of the only two genes significantly down-regulated out of the 1,176 genes analyzed stresses the hypothesis that FN may behave as an important metastasis suppressor gene in mammary cancer.

Differential gene expression profiles between tumor biopsies and short-term primary cultures of ovarian serous carcinomas: Identification of novel molecular biomarkers for early diagnosis and therapy

Objective. To identify novel molecular biomarkers useful for the early diagnosis and therapy of ovarian cancer by gene expression profiling. To compare the genetic fingerprints of flash-frozen ovarian serous carcinomas to those of matched highly purified primary tumor cell cultures. Methods. Gene expression profiles of 19 flash-frozen ovarian serous papillary carcinoma (OSPC) were analyzed and compared to 15 controls (highly purified human ovarian surface epithelium short-term cultures, HOSE) using oligonucleotide microarrays complementary to > 14,500 human genes. In addition, gene expression profiling of 5 highly purified primary OSPC cultured in vitro for less than 2 weeks was compared to flash-frozen ovarian carcinoma biopsies obtained from matched samples. Quantitative RT-PCR and IHC staining techniques were used to validate microarray data at RNA and protein levels for some of the differentially expressed genes. Results. Unsupervised analysis of gene expression data readily distinguished normal tissue from flash-frozen OSPC and identified 901 and 557 genes that exhibited >3-fold up-regulation or down-regulation, respectively, in OSPC when compared to HOSE. Mammaglobin 2, an ovarian secreted protein, was identified as the top differentially expressed gene in OSPC (19 out 19 OSPC versus 0 out of 15 HOSE) with over 827-fold up-regulation relative to HOSE. The claudin and kallikrein family of proteins including the clostridium perfringens enterotoxin receptors claudin 3 and 4, kallikreins 6, 7, 8, 10, 11 and the immunomodulatory molecule B7-H4 were found among the most highly overexpressed genes in OSPC when compared to HOSE. Genetic fingerprints of flash-frozen OSPC were found to have high correlation with those of purified primary OSPC short-term in vitro cultures with only 31 out of 8637 genes (0.35%) differentially expressed between the two groups. Conclusions. Short-term in vitro culture of primary ovarian carcinomas may greatly increase the purity of ovarian tumor RNA available for gene expression profiling without causing major alteration in OSPC fingerprints. Mammaglobin 2, kallikreins 6, 7, 8, 10, 11, claudin 3 and 4 and B7-H4 gene expression products represent candidate biomarkers endowed with great potential for early screening and therapy of OSPC patients.

Differential Targets and Subcellular Localization of Antitumor Alkyl-lysophospholipid in Leukemic Versus Solid Tumor Cells

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.

Topotecan and Paclitaxel in Previously Treated Patients with Relapsed Small Cell Lung Cancer: Phase II Trial of the North Central Cancer Treatment Group

Small cell lung cancer (SCLC) is one of the most aggressive and lethal cancers in humans. It constitutes approximately 15 to 25% of all cases of primary lung cancers.1 Initial response rates of 70 to 90% for both limited and extensive stages of SCLC may be achieved using standard combination cytotoxic chemotherapy agents. The 5-year survival for limited-state SCLC is 15 to 25%, but patients with extensivestage SCLC rarely survive 5 years. Even among patients who achieve a complete response, there is a high rate of relapse.2 Until recently, there has been no well-established treatment available for patients with recurrent SCLC.3 Topotecan is a camptothecin analogue that stabilizes the covalent adduct between topoisomerase I and DNA. In S-phase cells, these topo I-DNA adducts are converted into double-strand breaks that seem to be responsible for cytotoxicity.4 Several phase II5–8 trials as well as a phase III trial3 have demonstrated antitumor efficacy, manageable toxicities, and acceptable safety profile of topotecan in both chemo-naive and previously treated patients with SCLC. Patients in the topotecan group derived significant palliative benefits in general symptoms (e.g., anorexia, fatigue, interference with daily activities, and pulmonary symptoms) over CAV chemotherapy in a randomized trial.3 Although topotecan is currently approved for second-line therapy in SCLC at a starting dose of 1.5 mg/m by a daily 30-minute intravenous infusion for 5 consecutive days of a 21-day cycle, advanced age, extensive pretreatment, prior platinum therapy, prior radiotherapy, and renal impairment are potential risk factors for increased myelosuppression during topotecan therapy. Several studies suggest that the 1.0and 1.25-mg/m doses of topotecan may be appropriate for patients with such high-risk factors.9–11 Paclitaxel is a chemotherapeutic agent that promotes the assembly and stabilization of microtubules. Such binding causes cells to form abundant arrays of disorganized and dysfunctional microtubules, leading to apoptosis. Although there is documented single-agent activity of paclitaxel in phase II studies among chemo-naive patients with SCLC with extensive disease,12,13 response duration is short, which suggests that paclitaxel is not sufficient as a single agent. It is also widely accepted that combinations of cytotoxic drugs produce higher response rates and survival rates in patients with SCLC compared with single-agent therapy. The combination of topotecan and paclitaxel is rational as they have non-overlapping mechanisms of action and are both active agents in SCLC, as discussed previously. Moreover, there is demonstrable in vitro synergy of this combination in SCLC cell lines,14,15 although there are insufficient data on sequence-specific synergy. Phase II trials of the combination of topotecan and paclitaxel that have been reported used a 5-day topotecan schedule as first-line treatment in patients with extensive SCLC.16–19 A randomized phase II study evaluating four treatment combinations17 showed that paclitaxel (230 mg/m on day 1) plus topotecan (1 mg/m on days 1-5) produced excessive toxicity, with toxic deaths occurring in 25% of patients. Objective response rate was 54% with a median survival of 13.8 months. The toxicities were ameliorated in the treatment arm using an attenuated paclitaxel dose of 175 mg/m, with treatment-related death occurring in 3% of patients. Objective response rate in this arm was 69% with a median survival of 9.9 months and 1-year survival of 40%, similar to standard etoposide plus cisplatin chemotherapy. Another phase II trial in patients with chemo-naive extensive SCLC used topotecan 1 mg/m (first three patients received 1.25 mg/m) daily for 5 days and paclitaxel (135 mg/m) on day 5 via a 24-hour infusion every 4 weeks.16 Despite prophylactic G-CSF administration, this combination was associated with a high incidence of myelosuppression *Mayo Clinic and Mayo Foundation, Rochester, Minnesota, †Illinois Oncology Research Association CCOP, Peoria, Illinois; ‡Cedar Rapids Oncology Project CCOP, Cedar Rapids, Iowa; §Carle Cancer Center CCOP, Urbana, Illinois; Missouri Valley Cancer Consortium, Omaha, Nebraska; ¶Duluth CCOP, Duluth, Minnesota; **Wichita Community Clinical Oncology Program, Wichita, Kansas; ††Siouxland HematologyOncology Associates, Sioux City, Iowa. Address correspondence to: Grace K. Dy, MD, Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. E-mail: Dy.Grace@mayo.edu Copyright © 2006 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/06/0103-0211

Caveolin-1 Deficiency (−/−) Conveys Premalignant Alterations in Mammary Epithelia, with Abnormal Lumen Formation, Growth Factor Independence, and Cell Invasiveness

During breast cancer development, the luminal space of the mammary acinar unit fills with proliferating epithelial cells that exhibit growth factor-independence, cell attachment defects, and a more invasive fibroblastic phenotype. Here, we used primary cultures of mammary epithelial cells derived from genetically engineered mice to identify caveolin-1 (Cav-1) as a critical factor for maintaining the normal architecture of the mammary acinar unit. Isolated cultures of normal mammary epithelial cells retained the capacity to generate mammary acini within extracellular matrix. However, those from Cav-1 (-/-) mice exhibited defects in three-dimensional acinar architecture, including disrupted lumen formation and epidermal growth factor-independent growth due to hyperactivation of the p42/44 mitogen-activated protein kinase cascade. In addition, Cav-1-null mammary epithelial cells deprived of exogenous extracellular matrix underwent a spontaneous epithelial-mesenchymal transition, with reorganization of the actin cytoskeleton, and E-cadherin redistribution. Mechanistically, these phenotypic changes appear to be caused by increases in matrix metalloproteinase-2/9 secretion and transforming growth factor-beta/Smad-2 hyperactivation. Finally, loss of Cav-1 potentiated the ability of growth factors (hepatocyte growth factor and basic fibroblast growth factor) to induce mammary acini branching, indicative of a more invasive fibroblastic phenotype. Thus, a Cav-1 deficiency profoundly affects mammary epithelia by modulating the activation state of important signaling cascades. Primary cultures of Cav-1-deficient mammary epithelia will provide a valuable new model to study the spatial/temporal progression of mammary cell transformation.

A Trangenic Mouse Model of Plasma Cell Malignancy Shows Cytogenetic and Gene Expression Profiles Similar to Human Multiple Myeloma.

Multiple myeloma is an incurable plasma cell malignancy for which existing animal models are limited. Human plasma cell tumors are genetically diverse, with no single chromosomal abnormality defining the disease, however, dysregulation of the genes c-myc and bcl-xl are both commonly observed. We have previously shown that targeted expression of c-myc and bcl-xl transgenes in mouse plasma cells produces malignancy which displays features of human myeloma such as localization of tumor cells to the bone marrow and lytic bone lesions. Tumors are also present at extramedullary sites (Cheung et al., J. Clin. Invest. 113: 1763, 2004). Tumors rapidly develop (median 16 weeks) in 100% of mice, and can be adoptively transferred to syngeneic controls using as few as 1 million tumor cells to produce tumors in as few as 10 days. Adoptive transfer of similar cell numbers from younger double transgenic mice, without evidence of malignancy, results in increased tumor latency (>8 weeks) or the absence of tumor formation, suggesting that an accumulation of genetic changes is required for tumor development. In order to understand the specific genetic alterations required for tumor progression and for localization of tumors to the bone marrow vs extramedullary sites, we have undertaken a detailed analysis of plasma cell tumors in myc/bcl-xl mice and have begun to compare them with human multiple myeloma. Analysis of cell surface markers shows the majority of tumors have a plasmablast phenotype, expressing CD138+, B220+, CD38+, and CD19+. This result is confirmed by RT-PCR for B cell and plasma cell specific markers Pax5, Xbp1 and Blimp1, which can be detected in tumor samples. In addition, transcripts for Mip1α, EZH2, and Dusp6, genes shown to be upregulated in human myeloma, can also be detected in the mouse myc/bcl-xl tumors. Spectral karyotype analysis of metaphase chromosomes from primary tumor cell cultures demonstrates that a variety of chromosomal abnormalities are present in mouse tumors, including trisomies and translocations, similar to what is observed in human myeloma. The most frequently aberrant chromosomes are 12 and 16, followed by chromosomes 1 and 4. Interestingly, two common sites for translocations were identified; 12F which corresponds to the mouse immunoglobulin heavy chain locus, and 4D, which corresponds to a genomic region containing genes for plasma cell tumor susceptibility (Bliskovsky et al., PNAS 100:14982, 2003). Further characterization of these translocations are being done to identify the precise breakpoints involved, and analysis of gene expression by RT-PCR and microarray analysis will be correlated to specific chromosomal abnormalities. Additionally, global gene expression profiles from myc/bcl-xl tumor cell cultures have been compared to existing profiles of human myeloma (Zhan et al., Blood 99: 1745, 2002). Our preliminary comparison of gene expression profiles from myc/bcl-xl tumors to human myeloma tumors with high myc expression show the mouse tumors are more similar to human tumors than to normal plasma cells. These data suggest the myc/bcl-xl mouse tumors are similar to a subset of human myelomas, and will provide insight into the specific genes and pathways underlying human disease.

Functional tumor reactive T cells in bone marrow and blood of pancreatic cancer patients

4113 Background: Memory T-cells specifically reactive to tumor antigens should be an ideal source to generate therapeutic effector cells against pancreatic cancer. To our knowledge functionally active, specific memory T-cells have not been demonstrated in pancreatic cancer patients. To investigate if functional memory T-cells are present in the bone marrow (BM) of pancreatic cancer patients, we performed in vitro stimulation with tumor associated antigens presented by autologous dendritic cells (DC). Methods: DC and T-cells were generated in a 14-day culture with GM-CSF and IL-4 (DC) and/or IL-2, IL-4, IL-7 (TC) from BM derived CD34+ progenitor cells of 35 patients with pancreatic carcinoma and chronic pancreatitis. DC were pulsed with lysate from autologous tumor cells, Muc I peptide - fragments and PBMCs (peripheral blood mononuclear cells as negative controls). Tumor specific T-cell response was evaluated in a single-cell interferon-γ-, IL4-, IL10- and IL12- release immunospot assay (Elispot). Cytotoxicity of CD8+ T-cells against primary cultured tumor cells was measured by a functional DNA-release, and trypan-blue staining assay. Results: In the individual patient, T-cells stimulated by tumor lysate pulsed DC as well as DC pulsed with Muc I peptide showed a significantly higher response in the INF-γ Elispot assay compared with T-cells stimulated by PBMC-lysate pulsed DC. The tumor specific T-cell frequency found in the bone marrow was consistently high (49/105 to 109/105) in all patients compared with the response found in the periferal blood (9/105 to 53/105; p<0,05 to p<0,001). In the cytotoxicity assay, the response was significantly higher when T-cells were stimulated with tumor lysate pulsed DC compared with PBMC-lysate pulsed DC. Conclusions: Our data indicate for the first time a regular occurrence of tumor antigen specific T cell responses during the course of pancreatic cancer that lead to the generation and enrichment of functional tumor cell-reactive memory T cells in the bone marrow. The BM may therefore be considered an important lymphoid organ for the induction or maintenance of anti tumor immune responses in pancreatic cancer. No significant financial relationships to disclose.

Vascular endothelial growth factor in salivary pleomorphic adenomas: one of the reasons for their poorly vascularized stroma

To better understand the poorly vascularized background of the stroma of pleomorphic adenomas, we attempted to determine the expression of molecules related to blood vessels and hypoxic conditions in pleomorphic adenoma. Surgical specimens and tumor cells in primary culture of salivary pleomorphic adenomas were used for immunohistochemistry for CD31, vascular endothelial growth factor (VEGF) and its receptors Flk-1 and Flt-1, as well as for hypoxia markers, such as hypoxia-inducible factor-1alpha (HIF-1alpha) and lactate dehydrogenase-1 (LDH). At the same time, alternative splicing modes of the VEGF gene and expression levels of the HIF-1alpha gene were analyzed in surgical specimens by means of reverse-transcription polymerase chain reaction (RT-PCR) and direct sequencing of the PCR products. In addition to co-immunolocalization with CD31+ vascular endothelial cells, VEGF and its receptors were demonstrated in normal duct epithelial and myoepithelial cells as well as in tumor cells in ductal structures and in myxochondroid stromata. Immunolocalizations for HIF-1alpha and LDH were confirmed in the VEGF-positive area. Immunofluorescence signals for VEGF and others were confirmed in pleomorphic adenoma cells in culture. RT-PCR results showed that there were at least four splicing modes of the VEGF gene, among which VEGF(121) was most enhanced, and higher HIF-1alpha levels in pleomorphic adenomas. The results suggest that pleomorphic adenoma cells produce VEGF in several functional forms for their own proliferation or differentiation, and that the VEGF expression is controlled by hypoxic circumstances of poorly vascularized pleomorphic adenomas.

761: Antiproliferative and Pro-Apoptotic Effects of Mycobacterial Cell Wall-DNA Complex on Human Bladder Tumor Cells in Primary Culture

761: Antiproliferative and Pro-Apoptotic Effects of Mycobacterial Cell Wall-DNA Complex on Human Bladder Tumor Cells in Primary Culture

Functional endothelin ET B receptors are selectively expressed in human oligodendrogliomas

Endothelin-1 (ET-1), a vasoactive and mitogenic peptide mainly produced by vascular endothelial cells, may be involved in the progression of several human tumors. Here, we present an immunohistochemical analysis of the expression pattern of ET-1 receptor subtypes (ET A-R and ET B-R) and a functional study of their potential role in human oligodendrogliomas and oligoastrocytomas. By comparison, we assessed the corresponding expression patterns of glioblastomas. Interestingly, a nuclear localization of ET-1 receptor subtypes (associated or not with a cytoplasmic labeling) was constantly observed in tumor cells from all three glioma types. Moreover, we noted a distinct receptor distribution in the different gliomas: a nuclear expression of ET B-R by tumor cells was found to be restricted to oligodendrogliomas and oligoastrocytomas, while a nuclear expression of ET A-R was only detected in tumor cells from some glioblastomas. Using primary cultures of oligodendroglial tumor cells, we confirmed the selective expression of nuclear ET B-R, together with a plasma membrane expression, and further demonstrated that this receptor was functionally coupled to intracellular signaling pathways known to be involved in cell survival and/or proliferation: extracellular signal-regulated kinase and focal adhesion kinase activation, actin cytoskeleton reorganization. In addition, impairment of ET B-R activation in these cells by in vitro treatment with an ET B-R-specific antagonist induced cell death. These data point to ET-1 as a possible survival factor for oligodendrogliomas via ET B-R activation and suggest that ET B-R-specific antagonists might constitute a potential therapeutic alternative for oligodendrogliomas.

Associations and Interactions between Ets-1 and Ets-2 and Coregulatory Proteins, SRC-1, AIB1, and NCoR in Breast Cancer

Associations between p160 coactivator proteins and the development of resistance to endocrine treatment have been described. We hypothesized that nuclear receptor coregulatory proteins may interact with nonsteroid receptors. We investigated the mitogen-activated protein kinase-activated transcription factors, Ets, as possible interaction proteins for the coactivators SRC-1 and AIB1 and the corepressor NCoR in human breast cancer. Expression and coexpression of Ets and the coregulatory proteins was investigated using immunohistochemistry and immunofluorescence in a cohort of breast tumor patients (N = 134). Protein expression, protein-DNA interactions and protein-protein interactions were assessed using Western blot, electromobility shift, and coimmunoprecipitation analysis, respectively. Ets-1 and Ets-2 associated with reduced disease-free survival (P < 0.0292, P < 0.0001, respectively), whereas NCoR was a positive prognostic indicator (P < 0.0297). Up-regulation of Ets-1 protein expression in cell cultures derived from patient tumors in the presence of growth factors associated with tumor grade (P < 0.0013; n = 28). In primary breast tumor cell cultures and in the SKBR3 breast cell line, growth factors induced interaction between Ets and their DNA response element, induced recruitment of coactivators to the transcription factor-DNA complex, and up-regulated protein expression of HER2. Ets-1 and Ets-2 interacted with the coregulators under basal conditions, and growth factors up-regulated Ets-2 interaction with SRC-1 and AIB1. Coexpression of Ets-2 and SRC-1 significantly associated with the rate of recurrence and HER expression, compared with patients who expressed Ets-2 but not SRC-1 (P < 0.0001 and P < 0.0001, respectively). These data describe associations and interactions between nonsteroid transcription factors and coregulatory proteins in human breast cancer.

Variability in infectivity of primary cell cultures of human brain tumors with HSV-1 amplicon vectors

We investigated the variability in infectivity of cells in primary brain tumor samples from different patients using an HSV-1 amplicon vector. We studied the infectivity of HSV-1 amplicon vectors in tumor samples derived from neurosurgical resections of 20 patients. Cells were infected with a definite amount of HSV-1 amplicon vector HSV-GFP. Transduction efficiency in primary tumor cell cultures was compared to an established human glioma line. Moreover, duration of transgene expression was monitored in different tumor cell types. All primary cell cultures were infectable with HSV-GFP with variable transduction efficiencies ranging between 3.0 and 42.4% from reference human Gli36 Delta EGFR glioma cells. Transduction efficiency was significantly greater in anaplastic gliomas and meningiomas (26.7+/-17.4%) compared to more malignant tumor types (glioblastomas, metastases; 11.2+/-8.5%; P=0.05). To further investigate the possible underlying mechanism of this variability, nectin-1/HevC expression was analyzed and was found to contribute, at least in part, to this variability in infectability. The tumor cells expressed the exogenous gene for 7 to 61 days with significant shorter expression in glioblastomas (18+/-13 d) compared to anaplastic gliomas (42+/-24 d; P<0.05). Interindividual variability of infectivity by HSV-1 virions might explain, at least in part, why some patients enrolled in gene therapy for glioblastoma in the past exhibited a sustained response to HSV-1-based gene- and virus therapy. Infectivity of primary tumor samples from respective patients should be tested to enable the development of efficient and safe herpes vector-based gene and virus therapy for clinical application.

Tissue factor is regulated by epidermal growth factor in normal and malignant human endometrial epithelial cells

Tissue Factor (TF), the initiator of the extrinsic coagulation cascade, is overexpressed in a variety of cancers. TF is also expressed in normal human endometrium but little is known about its expression or regulation in endometrial cancer. We demonstrate herein that TF is expressed in the endometrial adenocarcinoma cell line Ishikawa. Furthermore, epidermal growth factor (EGF) induces a rapid and sustained increase in TF expression. Estradiol and progesterone had no effect on basal or EGF-induced TF expression in Ishikawa cells. In contrast to the pronounced and sustained upregulation at the protein level, EGF treatment elicited only a modest and transient increase in TF mRNA levels. This activity corresponded to the response observed from an exogenous TF promoter construct. However, the induction of TF was abrogated by cycloheximide as well as actinomycin-D, inhibitors or protein- and mRNA-synthesis, respectively, demonstrating that EGF mediates its effect through activation of the TF gene. Fractionation experiments showed that EGF increases TF presence in caveolin-I containing membrane fractions. Coagulation and invasion assays were used to explore the physiological implications of TF regulation. The results demonstrate that EGF-mediated induction of TF increases the procoagulant activity and invasive potential of Ishikawa cells. Furthermore, immunocytochemistry confirmed that TF is regulated by EGF in primary cultures of normal endometrial epithelial cells and malignant tumor cells. In conclusion, EGF-mediated upregulation of TF results in accumulation of this glycoprotein in caveolae-like membrane fractions and increased coagulative and invasive potential. Our results suggest that TF may play an integral role in endometrial carcinogenesis.

Inverse relationship between ER-beta and SRC-1 predicts outcome in endocrine-resistant breast cancer.

The oestrogen receptor (ER) interacts with coactivator proteins to modulate genes central to breast tumour progression. Oestrogen receptor is encoded for by two genes, ER-α and ER-β. Although ER-α has been well characterized, the role of ER-β as a prognostic indicator remains unresolved. To determine isoform-specific expression of ER and coexpression with activator proteins, we examined the expression and localisation of ER-α, ER-β and the coactivator protein steroid receptor coactivator 1 (SRC-1) by immunohistochemistry and immunofluorescence in a cohort of human breast cancer patients (n=150). Relative levels of SRC-1 in primary breast cultures derived from patient tumours in the presence of β-oestradiol and tamoxifen was assessed using Western blotting (n=14). Oestrogen receptor-β protein expression was associated with disease-free survival (DFS) and inversely associated with the expression of HER2 (P=0.0008 and P<0.0001, respectively), whereas SRC-1 was negatively associated with DFS and positively correlated with HER2 (P<0.0001 and P<0.0001, respectively). Steroid receptor coactivator 1 protein expression was regulated in response to β-oestradiol or tamoxifen in 57% of the primary tumour cell cultures. Protein expression of ER-β and SRC-1 was inversely associated (P=0.0001). The association of ER-β protein expression with increased DFS and its inverse relationship with SRC-1 suggests a role for these proteins in predicting outcome in breast cancer.

Factors influencing nitroxide reduction and cytotoxicity in vitro.

Nitroxides have been shown to be effective antioxidants, radiation protectors, and redox-active probes for functional electron paramagnetic resonance (EPR) imaging. More recently, the nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-N-oxyl (Tempol) has been shown to exert differential cytotoxicity to tumor compared with normal cell counterparts. Nitroxides are readily reduced in tissues to their respective hydroxylamines, which exhibit less cytotoxicity in vitro and do not provide radiation protection or an EPR-detectable signal for imaging. In order to better understand factors that influence nitroxide reduction, the rate of reduction of Tempol in mouse and human cell lines and in primary cultures of tumor cells was measured using EPR spectroscopy. Additionally, the cytotoxicity of high concentrations of Tempol and the hydroxylamine of Tempol (Tempol-H) was evaluated in wild-type and glucose-6-phosphate dehydrogenase (G6PD)-deficient Chinese hamster ovary cells. The results show that in general Tempol was reduced at a faster rate when cells were under hypoxic compared with aerobic conditions. Neither depletion of intracellular glutathione nor treatment of cells with sodium cyanide influenced Tempol reduction rates. G6PD-deficient cells were found to reduce Tempol at a significantly slower rate than wild-type cells. Likewise, Tempol-induced cytotoxicity was markedly less for G6PD-deficient cells compared with wild-type cells. Tempol-H exhibited no cytotoxicity to either cell type. Tempol-mediated cytotoxicity was enhanced by glutathione depletion and inhibition of 6-phosphogluconate dehydrogenase in wild-type cells, but was unaltered in G6PD-deficient cells. Collectively, the results indicate that while the bioreduction of Tempol can be influenced by a number of factors, the hexose monophosphate shunt appears to be involved in both nitroxide reduction as well as cytotoxicity induced by high levels of exposure to Tempol.

276. Evaluation of an Oncolytic Adenovirus: Efficacy Studies on Primary Human Tumor Cell Cultures and Safety Studies in Non-Human Primates

There is a great need for agents that target therapeutic agents, such as viral vectors, to prostate cancer cells. We have used phage display to identify short disulfide-constrained heptapeptides that bind specifically to the extracellular-portion of the Prostate Specific Membrane Antigen (PSMA), a type II integral membrane protein over-expressed on the surface of prostate cancer cells. Two separate phage display peptide strategies were applied, in parallel, to purified PSMA protein bound to two separate substrates. We reasoned that peptide sequences common to both substrate selections would be specific binders of PSMA. Additionally, the design allowed for stringent cross selections, where phage populations from one selection condition could be applied to the alternative substrate. These strategies resulted in a series of phage displayed peptides able to bind to PSMA by ELISA and direct binding assays, both with purified protein and in prostate cancer cells. PSMA specific cell binding is competitively inhibited by soluble, purified PSMA. The synthesized peptides are capable of binding PSMA protein and enhancing its carboxypeptidase enzymatic activity, suggesting protein folding stabilization. The discovery of these peptides provides the foundation for subsequent development of peptide targeted therapeutics against prostate cancer.

Uptake of meta-iodobenzylguanidine in neuroendocrine tumours is mediated by vesicular monoamine transporters.

The radio-iodinated noradrenaline analogue meta-iodobenzylguanidine (MIBG) can be used for scintigraphy and radiation therapy of neuroendocrine (NE). The aim of the present study was to study the importance of vesicular monoamine transporters (VMATs) for the uptake of 123I-MIBG in NE tumours. In nude mice, bearing the human transplantable midgut carcinoid GOT1, all organs and xenografted tumours accumulated 123I after i.v. injection of 123I-MIBG. A high concentration of 123I was maintained in GOT1 tumours and adrenals, which expressed VMATs, but rapidly decreased in all other tissues. In the VMAT-expressing NE tumour cell lines GOT1 and BON and in VMAT-expressing primary NE tumour cell cultures (carcinoids, n=4 and pheochromocytomas, n=4), reserpine significantly reduced the uptake of 123I-MIBG. The membrane pump inhibitor clomipramine had no effect on the uptake of 123I-MIBG in GOT1 and BON cells, but inhibited the uptake in one out of four primary carcinoid cell cultures and three out of four primary pheochromocytoma cell cultures. In conclusion, VMATs and secretory granules are of importance for the uptake and retention of 123I-MIBG in NE tumours. Information about the type and degree of expression of VMATs in NE tumours may be helpful in future to select patients suitable for radiation therapy with radio-iodinated MIBG.

Differential Ca2+ channel activity in human primary neuroendocrine tumor cell cultures from fore- and midgut tumors

Differential Ca2+ channel activity in human primary neuroendocrine tumor cell cultures from fore- and midgut tumors

Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside.

Central nervous system malignant neoplasias, in particular, glioblastoma multiforme (GBM) have defied all current therapeutic modalities. New therapies involving tumor targeting approach are being explored. This approach relies on the identification of unique or over-expressed cell surface receptors or antigens on tumor cells. In that regard, we have identified receptor for an immune regulatory cytokine, interleukin-13 (IL-13), which is over-expressed on human malignant glioma cell lines and primary tumor cell cultures. To target IL-13 receptors (IL-13R) for cancer therapy, we have developed a recombinant fusion protein composed of IL-13 and a mutated form of Pseudomonas exotoxin (IL13-PE38QQR or IL-13 cytotoxin). The IL-13 cytotoxin was found to be highly selective and potent in killing human GBM cells in vitro while normal cells including immune cells, endothelial cells and normal brain cells were generally spared the cytotoxic effect of IL-13 cytotoxin. This is because these cells either expressed none or expressed low levels of IL-13R. Consistent with in vitro cytotoxic activity, IL-13 cytotoxin mediated remarkable anti-tumor activity to human glioma in animal xenograft models. The direct injection of IL-13 cytotoxin into subcutaneous human GBM tumors grown in nude mice produced complete and durable regression of established tumors. Intravenous and intraperitoneal administration of IL-13 cytotoxin also reduced tumor burden significantly with fewer complete responders. All animals tolerated therapy well with minimal toxicity to vital organs. Pre-clinical safety and toxicity studies were performed in mice, rats and monkeys. Systemic administration of IL-13 cytotoxin appeared to be well tolerated at high doses (up to 50 microg/kg). Intrabrain parenchyma administration of IL-13 cytotoxin at doses up to 100 microg/ml was very well tolerated without any evidence of gross or microscopic necrosis, whereas at 500 microg/ml dose, localized necrosis was observed in normal rat brain. Based on these encouraging pre-clinical studies, three Phase I/II clinical trials in adults with malignant glioma have been initiated. The first clinical trial involves convection-enhanced delivery (CED) of IL-13 cytotoxin into recurrent malignant glioma. This route of IL-13 cytotoxin administration appears to be fairly well tolerated with no neurotoxicity. The second clinical trial involves infusion of IL-13 cytotoxin by CED following tumor resection. The initial stage of the second study assessed histologic effect of drug administered prior to resection. In third one, IL-13 cytotoxin is infused by CED followed by tumor resection. All three clinical trials are currently ongoing.