Essential role of IFNbeta and CD38 in TNFalpha-induced airway smooth muscle hyper-responsiveness.

We recently identified autocrine interferon (IFN)beta as a novel mechanism mediating tumor necrosis factor (TNF)alpha-induced expression of inflammatory genes in airway smooth muscle (ASM) cells, including CD38, known to regulate calcium signaling. Here, we investigated the putative involvement of IFNbeta in regulating TNFalpha-induced airway hyper-responsiveness (AHR), a defining feature of asthma. Using our pharmacodynamic model to assess ex vivo AHR isolated murine tracheal rings, we found that TNFalpha-induced enhanced contractile responses to carbachol and bradykinin was abrogated by neutralizing anti-IFNbeta antibody or in tracheal rings deficient in CD38. In cultured human ASM cells, where CD38 has been involved in TNFalpha-induced enhanced calcium signals to carbachol and bradykinin, we found that neutralizing anti-IFNbeta prevented TNFalpha enhancing action only on carbachol responses but not to that induced by bradykinin. In a well-characterized model of allergic asthma (mice sensitized and challenged with Aspergillus fumigatus (Af)), we found heightened expression of both IFNbeta and CD38 in the airways. Furthermore, allergen-associated AHR to methacholine, assessed by lung resistance and dynamic compliance, was completely suppressed in CD38-deficient mice, despite the preservation of airway inflammation. These data provide the first evidence that ASM-derived IFNbeta and CD38 may play a significant role in the development of TNFalpha-associated AHR.

Human myocardial cell lines generated with SV40 temperature-sensitive mutant tsA58.

Conditionally transformed human myocardial cell lines would be a valuable resource for studying human cardiac cell biology. We generated clonal human fetal cardiocyte cell lines by transfection of fetal ventricular cardiac cell clones with a plasmid containing a replication-defective mutant of the temperature-sensitive SV40 strain tsA58. Multiple resulting cell lines showed similar features, namely: (1) T antigen (TAg) expression at both permissive (34 degrees C) and restrictive (40.5 degrees C) temperatures; (2) extended growth capacity in comparison with parental wild type, when grown at the permissive temperature; (3) both temperature-dependent and serum-responsive growth, and; (4) an incompletely differentiated fetal phenotype which was similar at both permissive and restrictive temperatures and in the presence and absence of serum. The transformed myocyte phenotype was demonstrated using immunocytochemistry, Western and Northern blotting, and reverse transcription-polymerase chain reaction (RT-PCR). Cell lines expressed skeletal alpha-actin, atrial natriuretic peptide (ANP), and keratins, but no sarcomeric myosin heavy chain or desmin. Immunoreactive sarcomeric actin was expressed predominantly as a truncated protein of approximately 38 kD. The phenotype of the transformed cells differs from that of the wild-type parental cells as well as from those reported by others who have used TAg to immortalize rodent or human ventricular myocytes. Our cell lines should provide a useful tool for study of the molecular mechanisms regulating growth and differentiation in human cardiac muscle cells.

Long-term follow-up of patients with malignant pleural mesothelioma receiving high-dose adenovirus herpes simplex thymidine kinase/ganciclovir suicide gene therapy.

PURPOSE: Delineation of the long-term follow-up data on a series of patients with malignant mesothelioma, who received a single intrapleural dose of a nonreplicative adenoviral (Ad) vector encoding the herpes simplex virus thymidine kinase "suicide gene" (Ad.HSVtk) in combination with systemic ganciclovir. EXPERIMENTAL DESIGN: This report focuses on the 21 patients receiving "high-dose" therapy, defined by an intrapleural dose of vector (> or =1.6 x 10(13) viral particles), where transgene-encoded tk protein was reliably identified on immunohistochemical staining. In 13 patients, the vector was deleted in the E1 and E3 regions of the Ad; in the other eight patients, the vector had deletions in the Ad genes E1 and E4. Safety, immunologic responses, transgene expression, and clinical responses were evaluated. RESULTS: Both the E1/E3-deleted vector and the E1/E4-deleted vector were well tolerated and safe, although production of the E1/E4 vector was more difficult. Posttreatment antibody responses against the tumors were consistently seen. Interestingly, we observed a number of clinical responses in our patients, including two long-term (>6.5 year) survivors, both of whom were treated with the E1/E4-deleted vector. CONCLUSIONS: Intrapleural Ad.HSVtk/ganciclovir is safe and well tolerated in mesothelioma patients and resulted in long-term durable responses in two patients. Given the limited amount of gene transfer observed, we postulate that Ad.HSVtk may have been effective due to induction of antitumor immune responses. We hypothesize that approaches aiming to augment the immune effects of Ad gene transfer (i.e., with the use of cytokines) may lead to increased numbers of therapeutic responses in otherwise untreatable pleural malignancies.

Treatment of lung cancer using clinically relevant oral doses of the cyclooxygenase-2 inhibitor rofecoxib: potential value as adjuvant therapy after surgery.

OBJECTIVE: To investigate the uses and limitations of cyclooxygenase- (COX) 2 inhibition using clinically relevant doses of oral rofecoxib in the treatment of murine models of non-small-cell lung cancer (NSCLC). SUMMARY BACKGROUND DATA: Overexpression of COX-2 has been reported in lung cancer. Several studies have demonstrated that high doses of COX-2 inhibitors could inhibit the growth of rodent and human lung cancer cell lines. The potential uses and limitations of COX-2 inhibition at doses equivalent to those currently approved for use in humans have not been well studied. METHODS: Three murine NSCLC cell lines were injected into the flanks of mice to establish tumor xenografts. Mice were treated orally with low doses of a COX-2 inhibitor (rofecoxib chow, 0.0075%). Mechanisms were evaluated by analysis of tumor-infiltrating lymphocytes. To study rofecoxib as adjuvant therapy, large established tumors (14-18 days after tumor inoculation) were surgically debulked and animals were treated with rofecoxib starting 3 days before surgery. Recurrence of the tumor after debulking was monitored. RESULTS: Rofecoxib significantly slowed the growth of small (0-120 mm) tumors (P < 0.01-0.05) in all 3 cell lines, with higher efficacy in the more immunogenic tumors. Minimal responses were noted in larger tumors. Rofecoxib appeared to augment CD8 T cell infiltration in immunogenic tumors. Rofecoxib significantly reduced the recurrence rate after debulking (P < 0.01). CONCLUSIONS: Clinically relevant doses of the COX-2 inhibitor rofecoxib given orally were effective in inhibiting the growth of small (but not large) tumors in 3 murine NSCLC cell lines tested and in preventing recurrences after surgical debulking. Depending on the immunogenicity of human tumors, COX-2 inhibition might be useful as adjuvant therapy for surgically resectable NSCLC.

Fetal liver as a source of autologous progenitor cells for perinatal tissue engineering.

Mesenchymal progenitor cells, isolated from adult bone marrow, have been shown to have utility for autologous tissue engineering. The possibility of isolating from the fetal hematopoietic system a cell population with similar potential, which could be used for autologous reconstruction of prenatally diagnosed congenital anomalies, has not been explored to date. Liver stromal cells isolated from a portion of the right lateral hepatic lobe of midgestation fetal lambs were expanded in vitro. Passage 1 cells displayed a uniform fibroblast-like morphology but could be induced to differentiate into skeletal muscle, adipocytes, chondrocytes, and endothelial cells by selective medium supplementation. By manipulating the extracellular matrix in vitro, spontaneously contracting cardiac myocyte-like cells could be generated as well. Multilineage differentiation was confirmed by morphology, protein expression, and upregulation of lineage-specific mRNA. The potential for engineering myocardial tissue was then investigated by transplanting early-passage progenitor cells, organized on a three-dimensional matrix, into the ventricle of an immunocompromised rat utilizing a previously described model of left ventricular tissue engineering. Survival, incorporation into the host myocardium, and cardiomyocytic differentiation of the transplanted cells were confirmed. We have demonstrated that mesenchymal progenitor cells with multilineage potential can be isolated from the fetal liver and have potential utility for autologous tissue engineering.

Differentially expressed apoptotic genes in early stage lung adenocarcinoma predicted by expression profiling.

OBJECTIVE: In undiseased lung epithelial cells, apoptosis is an evolutionarily conserved and genetically regulated form of cell suicide which plays an important role in development and in the maintenance of tissue homeostasis. Neoplastic lung cells develop the ability to deregulate growth by alterations in these genes which control apoptosis. Genomic profiling was used to compare gene expression levels in early stage lung adenocarcinomas and nonneoplastic pulmonary tissue in order to comprehensively identify alterations in the process of apoptosis. METHODS: RNA extracted from node negative, poorly differentiated lung adenocarcinomas (15 patients) and nonneoplastic pulmonary tissue (5 patients) was hybridized to oligonucleotide microarray filters containing 44,363 genes. Ontological classification was used to extract genes involved with apoptosis. Further analysis discovered a subset of differentially expressed genes for further study. RESULTS: Of the 308 apoptotic genes on the microarray filters, 24 genes were predicted to be differentially expressed in lung adenocarcinomas. Alterations in several genes (i.e., Akt, BcL-xL, PTEN, FAS) are consistent with the literature. We also identified 10 novel genes that have not been described in nonsmall cell lung cancer (i.e., RIP, Caspase 1, PDK-1). CONCLUSIONS: These results identified several potential apoptotic genes altered in lung cancer.

Gene expression profiling of malignant mesothelioma.

PURPOSE: Malignant mesothelioma is a uniformly fatal cancer of the pleural and peritoneal spaces. Several challenging clinical problems include poor understanding of the pathophysiology, inaccurate diagnosis from tissue samples, and unsuccessful treatment strategies. The purpose of this study was to use microarray analysis to identify specific gene expression changes in mesothelioma compared with normal mesothelium. EXPERIMENTAL DESIGN: We performed gene expression analysis on mesothelioma tissue specimens from 16 patients and compared these to 4 control pleural tissue samples using cDNA microarray filters with 4132 clones. Multiple normalization and analysis approaches were used. Quantitative reverse transcription-PCR and immunohistochemistry were used to validate results. RESULTS: Genes (166) were significantly up-regulated, and 26 were down-regulated. Validation of 18 genes using real-time PCR confirmed array predictions in every case. Analysis revealed activation of several key pathways including genes involved in glucose metabolism, mRNA translation, and cytoskeletal remodeling. Expression profiling identified processes likely responsible for 18-fluoro-2-deoxy-glucose uptake and tumor localization by positron emission tomography, and a role for hypoxia-inducible factor-1 was suggested. Potentially important up-regulated genes included gp96, lung resistance-related protein, galectin-3 binding protein, the M(r) 67,000 laminin receptor (on tumor vessels), and voltage-dependent anion channels. Prospective testing using reverse transcription-PCR confirmed up-regulation of these novel markers. CONCLUSIONS: Expression profiling revealed marked up-regulation of energy, protein translation, and cytoskeletal remodeling pathways in mesothelioma. Additional genes that could be important in our understanding of the pathogenesis of mesothelioma, aiding in diagnosis, or improving targets for therapy were also identified.

Alterations in cell cycle genes in early stage lung adenocarcinoma identified by expression profiling.

In normal lung epithelial cells, cellular division is an ordered, tightly regulated process involving multiple checkpoints that assess extracellular growth signals, cell size, and DNA integrity. In contrast, neoplastic lung cells develop the ability to bypass several of these checkpoints, particularly at the G1/S and G2/M boundaries. We used genomic profiling to compare gene expression levels in early stage lung adenocarcinomas and non-neoplastic pulmonary tissue in order to comprehensively identify alterations in the process of cell cycling. RNA extracted from node negative, poorly differentiated lung adenocarcinomas (15 patients) and non-neoplastic pulmonary tissue (5 patients) was hybridized to oligonu-cleotide microarray filters containing 44,363 genes. Ontological classification was used to extract genes involved with cell cycle progression. Further analysis discovered a subset of differentially expressed genes for further study. Of the 624 cell cycle genes on the microarray filters, 40 genes were predicted to be differentially expressed in lung adeno-carcinomas. Alterations in several genes (i.e., cyclin B1, cyclin D1, p21, MDM2) are consistent with published data in the literature. We also identified 19 novel genes that have neither been described in non-small cell lung cancer (i.e., cdc2, cullin 4A, ZAC, p57, DP-1, GADD45, PISSLRE, cdc20) nor in any other tumors (i.e., cyclin F, cullin 5, p34). These results identified several potential cell cycle genes altered in lung cancer.

Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing.

The Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors and for the ablation of unwanted transfused stem cells in recent clinical trials. With the aim of improving this therapeutic system, we compared the potential efficacy of adenoviral (Ad) vectors expressing enhanced tk mutants in vitro and in vivo. The previously created HSV-tk mutants dm30 and sr39, created by random sequence mutagenesis, were inserted into a standard Ad.RSV E1(-)E3(-) backbone using homologous recombination. GCV killing of Ad.HSV-tk, Ad.dm30-tk and Ad.sr39-tk was assessed in various tumor cell lines with a cell proliferation assay. Cells expressing the two TK mutants were two-to-five-fold more sensitive to GCV when compared with Ad.HSV-tk transduced cells in all cell lines tested (five human mesotheliomas, one human lung cancer, a human cervical carcinoma, a mouse fibrosarcoma, and a rat glioma line) at equal TK expression levels. Flank tumor models, including cell-mixing studies, assessed the in vivo efficacy of the engineered viruses in BALB/C and SCID mice. In all animal studies, Ad.dm30-tk and Ad.sr39-tk showed more tumor growth inhibition than Ad.HSV-tk when GCV was administered. The use of adenovirus-mediated gene transfer of both tk mutants dm30-tk and sr39-tk for cancer suicide gene therapy should provide a more effective and safer alternative to wild-type HSV-tk.

An E2F-responsive replication-selective adenovirus targeted to the defective cell cycle in cancer cells: potent antitumoral efficacy but no toxicity to normal cell.

To improve the transduction and distribution of adenovirus in a tumor mass, we generated an adenovirus to selectively replicate in tumors. We hypothesized that after infection the replicating adenovirus would spread throughout the tumor mass and cause direct oncolysis of tumor cells. E2F transcription factors are critical regulators of cell growth and are often overexpressed in cancer cells because of the frequent aberrations in the pRb/E2F/p16(INK4a) pathway. As a result, a majority of tumor cells exist in a high proliferative state. E2F-1 is a transcription factor that activates its own transcription and that of other genes involved in the G(1) to S transition phase of the cell cycle. We constructed an adenovirus (Ad(E2F-1(RC)) so that E1A expression and viral replication were under the control of the human E2F-1 promoter element. AdE2F-1(RC) virus replicated as efficiently as the wild-type adenovirus and caused extensive cell killing in a panel of tumor cells in vitro. In contrast, nonproliferating normal epithelial, fibroblast, and endothelial cells, which express no E2F-1, were not able to support AdE2F-1(RC) replication. In animal studies, different dosing regimens of AdE2F-1(RC) administered to flank xenografts of ovarian and lung cancers led to a significant therapeutic advantage often surpassing that seen in animals treated with the wild-type adenovirus. This novel selectively replicating adenovirus offers a promising treatment platform for a variety of cancers of which the hallmark is uncontrolled cell growth.

Replication-selective herpes simplex virus type 1 mutant therapy of cervical cancer is enhanced by low-dose radiation.

Herpes simplex virus type 1 (HSV-1)-based oncolytic treatment is a promising therapeutic approach for malignancy. Recombinant strains of HSV-1 containing mutations in the ICP 34.5 protein have been shown to replicate preferentially in rapidly proliferating malignant cells, resulting in a direct cytolytic effect. We assessed the efficacy of multimutated HSV-1 strains on human cervical cancer, and then used these viruses in combination with radiation therapy, the standard treatment for cervical cancer. The HSV-1 mutants 4009, 7020, 3616, and G207 induced significant lysis of three established human cervical cancer cell lines in vitro in a dose-dependent manner. G207 intratumoral treatment of established subcutaneous C33a tumors in severe combined immunodeficient (SCID) mice significantly reduced tumor burden by 50%. Weekly and triweekly treatments improved efficacy and inhibited flank tumor growth in an administration frequency-dependent manner without toxicity. Combination therapy of a low dose of radiation (1.5 or 3 Gy) and replication-selective HSV mutants infection exhibited increased antitumor effects against cervical cancer cells in vitro. The in vivo effect of G207 combined with low-dose radiation was studied in Me180 xenografts in athymic mice. Treatment of established Me180 tumor nodules with 3 Gy followed by intratumoral G207 administration greatly improved efficacy, resulting in 42% complete eradication of tumor. In conclusion, single and multiple intratumoral injections of G207 significantly reduced tumor burden in xenogeneic models of cervical cancer, and the addition of low-dose radiation further potentiated the effect. These results suggest that replication-selective HSV-1 mutants may be potent oncolytic agents for the treatment of cervical cancer.