Monica Gonzales

Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR.

Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for beta-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.

Identification and functional characterization of neo-poly(A) polymerase, an RNA processing enzyme overexpressed in human tumors.

ABSTRACT Poly(A) polymerase (PAP) plays an essential role in polyadenylation of mRNA precursors, and it has long been thought that mammalian cells contain only a single PAP gene. We describe here the unexpected existence of a human PAP, which we call neo-PAP, encoded by a previously uncharacterized gene. cDNA was isolated from a tumor-derived cDNA library encoding an 82.8-kDa protein bearing 71% overall similarity to human PAP. Strikingly, the organization of the two PAP genes is nearly identical, indicating that they arose from a common ancestor. Neo-PAP and PAP were indistinguishable in in vitro assays of both specific and nonspecific polyadenylation and also endonucleolytic cleavage. Neo-PAP produced by transfection was exclusively nuclear, as demonstrated by immunofluorescence microscopy. However, notable sequence divergence between the C-terminal domains of neo-PAP and PAP suggested that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP did not show evidence of phosphorylation on Western blot analysis, which was unexpected in the context of a conserved cyclin recognition motif and multiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intriguingly, Northern blot analysis demonstrated that each PAP displayed distinct mRNA splice variants, and both PAP mRNAs were significantly overexpressed in human cancer cells compared to expression in normal or virally transformed cells. Neo-PAP may therefore be an important RNA processing enzyme that is regulated by a mechanism distinct from that utilized by PAP.

Role of Cripto-1 in Stem Cell Maintenance and Malignant Progression

Cripto-1 is critical for early embryonic development and, together with its ligand Nodal, has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Like other embryonic genes, Cripto-1 performs important roles in the formation and progression of several types of human tumors, stimulating cell proliferation, migration, epithelial to mesenchymal transition, and tumor angiogenesis. Several studies have demonstrated that cell fate regulation during embryonic development and cell transformation during oncogenesis share common signaling pathways, suggesting that uncontrolled activation of embryonic signaling pathways might drive cell transformation and tumor progression in adult tissues. Here we review our current understanding of how Cripto-1 controls stem cell biology and how it integrates with other major embryonic signaling pathways. Because many cancers are thought to derive from a subpopulation of cancer stem-like cells, which may re-express embryonic genes, Cripto-1 signaling may drive tumor growth through the generation or expansion of tumor initiating cells bearing stem-like characteristics. Therefore, the Cripto-1/Nodal signaling may represent an attractive target for treatment in cancer, leading to the elimination of undifferentiated stem-like tumor initiating cells.

Lentivirus vector-mediated expression of tumor-associated epitopes by human antigen presenting cells

Directing the human immune system to recognize and eliminate tumor cells is the ultimate goal of cancer immunotherapy. Vaccinating patients with autologous antigen presenting cells (APC) expressing tumor-associated antigens (TAA) represents a promising approach for activating tumor-reactive T cells in vivo. In addition, APC expressing TAA provide a means of generating tumor-specific T cells in vitro, for therapeutic and diagnostic applications. Lentiviral vectors are attractive vehicles for introducing TAA-encoding genes into APC. In this study, lentiviral vectors expressing the reporter gene GFP or the melanoma-associated antigen tyrosinase were used to transduce three different kinds of human APC: monocyte-derived dendritic cells (DC), CD40L-activated B lymphocytes, and Epstein Barr virus (EBV)-transformed B lymphocytes. Using optimized transduction conditions for each cell type, tyrosinase was expressed at levels sufficient to stimulate antigen-specific major histocompatibility complex (MHC) class I-restricted T cells from melanoma patients. While transduced EBV-B cells demonstrated the highest level of transgene expression, optimal T-cell recognition was achieved with transduced DC. Substituting the CAG promoter for PGK in lentiviral constructs enhanced transgene expression in DC and EBV-B cells, amplifying T cell recognition. Lentiviruses inducing sustained transgene expression with relatively low cellular toxicity and background viral gene expression may be ideal vectors for immunotherapeutic applications.

CD4+ T-Cell Recognition of Mutated B-RAF in Melanoma Patients Harboring the V599E Mutation

The potential of antigen-directed cancer immunotherapy has not been fully realized, perhaps because many commonly targeted tumor associated proteins are not essential to maintaining the malignant cell phenotype. A constitutively activating mutation in the signaling molecule BRAF is expressed frequently in melanomas and may play an important role in the biology of this disease. A 29-mer B-Raf peptide incorporating the V599E mutation was used for in vitro stimulation of lymphocytes derived from melanoma patients, generating MHC class II-restricted CD4+ T cells specific for this peptide as well as for melanoma cells expressing B-Raf V599E. Mutated B-Raf exemplifies targets that may be ideal for immunotherapy.

Cripto-1 Is Required for Hypoxia to Induce Cardiac Differentiation of Mouse Embryonic Stem Cells

Cripto-1 is a membrane-bound protein that is highly expressed in embryonic stem cells and in human tumors. In the present study, we investigated the effect of low levels of oxygen, which occurs naturally in rapidly growing tissues, on Cripto-1 expression in mouse embryonic stem (mES) cells and in human embryonal carcinoma cells. During hypoxia, Cripto-1 expression levels were significantly elevated in mES cells and in Ntera-2 or NCCIT human embryonal carcinoma cells, as compared with cells growing with normal oxygen levels. The transcription factor hypoxia-inducible factor-1alpha directly regulated Cripto-1 expression by binding to hypoxia-responsive elements within the promoter of mouse and human Cripto-1 genes in mES and NCCIT cells, respectively. Furthermore, hypoxia modulated differentiation of mES cells by enhancing formation of beating cardiomyocytes as compared with mES cells that were differentiated under normoxia. However, hypoxia failed to induce differentiation of mES cells into cardiomyocytes in the absence of Cripto-1 expression, demonstrating that Cripto-1 is required for hypoxia to fully differentiate mES cells into cardiomyocytes. Finally, cardiac tissue samples derived from patients who had suffered ischemic heart disease showed a dramatic increase in Cripto-1 expression as compared with nonischemic heart tissue samples, suggesting that hypoxia may also regulate Cripto-1 in vivo.

Growth factor induction of cripto-1 shedding by glycosylphosphatidylinositol-phospholipase D and enhancement of endothelial cell migration

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

Requirement of Glycosylphosphatidylinositol Anchor of Cripto-1 for trans Activity as a Nodal Co-receptor

Cripto-1 (CR-1) has an indispensable role as a Nodal co-receptor for patterning of body axis in embryonic development. CR-1 is reported to have a paracrine activity as a Nodal co-receptor, although CR-1 is primarily produced as a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Regulation of cis and trans function of CR-1 should be important to establish the precise body patterning. However, the mechanism by which GPI-anchored CR-1 can act in trans is not well known. Here we confirmed the paracrine activity of CR-1 by fluorescent cell-labeling and immunofluorescent staining. We generated COOH-terminal-truncated soluble forms of CR-1 based on the attachment site for the GPI moiety (ω-site), which we identified in the present study. GPI-anchored CR-1 has a significantly higher activity than COOH-terminal-truncated soluble forms to induce Nodal signal in trans as well as in cis. Moreover, transmembrane forms of CR-1 partially retained their ability to induce Nodal signaling only when type I receptor Activin-like kinase 4 was overexpressed. NTERA2/D1 cells, which express endogenous CR-1, lost the cell-surface expression of CR-1 after phosphatidylinositol-phospholipase C treatment and became refractory to stimulation of Nodal. These observations suggest that GPI attachment of CR-1 is required for the paracrine activity as a Nodal co-receptor.

Evaluation of Prime/Boost Regimens Using Recombinant Poxvirus/Tyrosinase Vaccines for the Treatment of Patients with Metastatic Melanoma

Purpose: Two clinical trials were conducted to evaluate the clinical efficacy and immunologic impact of vaccination against the tyrosinase protein plus systemic interleukin 2 (IL-2) administration in patients with advanced metastatic melanoma. Experimental Design: Full-length tyrosinase was employed as an immunogen to induce diverse immunologic responses against a commonly expressed melanoma antigen. Heterologous prime/boost vaccination with recombinant vaccinia and fowlpox vectors encoding tyrosinase was first explored in a randomized three-arm phase II trial, in which vaccines were administered alone or concurrently with low-dose or high-dose IL-2. In a subsequent single cohort phase II trial, all patients received the same vaccines and high-dose IL-2 sequentially rather than concurrently. Results: Among a total of 64 patients treated on these trials, 8 objective partial responses (12.5%) were observed, all in patients receiving high-dose IL-2. Additional patients showed evidence of lesional regression (mixed tumor response) or overall regression that did not achieve partial response status (minor response). In vitro evidence of enhanced immunity against tyrosinase following protocol treatments was documented in 3 of 49 (6%) patients tested serologically, 3 of 23 (13%) patients tested for T-cell recognition of individual tyrosinase peptides, and 4 of 16 (25%) patients tested for T-cell recognition of full-length tyrosinase protein with real-time reverse transcription-PCR techniques. Conclusions: Whereas prime/boost immunization with recombinant vaccinia and fowlpox viruses enhanced antityrosinase immunity in some patients with metastatic melanoma, it was ineffective alone in mediating clinical benefit, and in combination with IL-2 did not mediate clinical benefit significantly different from that expected from treatment with IL-2 alone.

Cripto‐1 Is a Cell Surface Marker for a Tumorigenic, Undifferentiated Subpopulation in Human Embryonal Carcinoma Cells

Deregulation of stem cells is associated with the generation and progression of malignant tumors. In addition, genes that are associated with early embryogenesis are frequently expressed in cancer. Cripto‐1 (CR‐1), a glycosylphosphatidylinositol‐linked glycoprotein, is expressed during early embryogenesis and in various human carcinomas. We demonstrated that human embryonal carcinoma (EC) cells are heterogeneous for CR‐1 expression and consist of two distinct subpopulations: a CR‐1High and a CR‐1Low population. By segregating CR‐1High and CR‐1Low populations of NTERA2/D1 EC cells by fluorescence‐activated cell sorting, we demonstrated that CR‐1High cells were more tumorigenic than CR‐1Low cells by an in vitro tumor sphere assay and by in vivo xenograft formation. The CR‐1High population was enriched in mRNA expression for the pluripotent embryonic stem (ES) cell genes Oct4, Sox2, and Nanog. CR‐1 expression in NTERA2/D1 cells was regulated by a Smad2/3‐dependent autocrine loop, by the ES cell‐related transcription factors Oct4/Nanog, and partially by the DNA methylation status of the promoter region. These results demonstrate that CR‐1 expression is enriched in an undifferentiated, tumorigenic subpopulation and is regulated by key regulators of pluripotent stem cells. STEM CELLS 2010;28:1303–1314