Functional characterization of hepatoma‐specific stem cell antigen‐2

Abstract

Identification of tumor-specific antigens and genetic pathways may lead to potential diagnostic and therapeutic applications in cancer treatment. cDNA microarray has been used in cancer gene profiling, but the broad spectrum of data accruing and narrow signal-to-noise range of this technology have limited its use in rapid identification of highly differentially expressed tumor genes. Here, we used a modified suppression subtractive hybridization (SSH) method to isolate a small number of highly differentially expressed genes from murine hepatoma cells. For functional analysis of these hepatoma-specific genes, we employed the small interference RNA (siRNA)-mediated gene silencing method with lentiviral vectors, which have the advantages of high delivery efficiency and long lasting effect. Stem cell antigen-2 (Sca-2) was identified as one of the highest differentially expressed tumor antigens. Lentiviral siRNA successfully suppressed >90% of Sca-2 expression and the suppression lasted longer than 3 mo. Interestingly, inhibition of Sca-2 induced rapid hepatoma cell apoptosis, and the survival Sca-2-negative hepatoma cells exhibited high sensitivity to extrinsic tumor necrosis factor alpha (TNF-a) apoptosis signal but not intrinsic apoptosis signal. Analysis of TNF receptor 1 (TNFR1) by flow cytometry and Western blotting indicated that Sca-2 expression downregulated cell surface but not de novo synthesis of TNFR1 in the hepatoma cells. Together, our results suggested that Sca-2 was a signal transducer situated at the nexus of surface molecules regulating death receptor-mediated apoptosis. The technology illustrated that this method can deduce a small number of highly differentially expressed tumor genes that may have diagnostic and therapeutic potential. ß 2004 Wiley-Liss, Inc.

An effective cancer vaccine modality: Lentiviral modification of dendritic cells expressing multiple cancer-specific antigens

“…The actual transduction efficiency is presumably higher than that detected by flow cytometry based on our previous assay using a sensitive reporter gene, placenta alkaline phosphatase [21]. Sca-2 expression was the highest of the three TAAs in the 1MEA7R hepatoma [23]. To compare the expression levels in BMDCs, we transduced immature DCs with LV-Sca-2, LV-GP38, LV-RABP1 and control LV-nLacZ, and RNA was harvested 48 h later and analyzed by semi-quantitative RT-PCR.…”

“…Such danger signals can be in the form of bacterial toxins or viral RNAs. In this study, we modified dendritic cells with LVs that encode multiple cancer antigens; these are highly differentially expressed hepatoma antigens identified previously by suppression subtractive hybridization cDNA cloning [23]. Combining the three hepatoma antigens, Sca-2, GP38, and RABP1, we demonstrated an effective anti-cancer immunity using modified dendritic cells.…”

“…To explore a novel dendritic cell (DC) cancer vaccine strategy, we cloned into lentiviral vectors the full-length cDNAs of three highly differentially expressed tumor-associated antigens that have been previously identified using an improved suppression subtractive hybridization cDNA cloning method [23]. Mouse bone marrow-derived dendritic cells (BMDCs) were cultured and transduced with the three LVs as illustrated in Fig.…”

“…Therefore, novel therapeutic strategies and a better understanding of HCC immunobiology are urgently required. Using murine hepatoma 1MEA7R cell line and its parental hepatocyte cell line BNL.CL2 as a model system, we have applied a modified suppression subtractive hybridization (SSH) cDNA screening method and identified a few highly differentially expressed murine hepatoma antigens; among the highest expressed tumor-associated antigens (TAAs) include stem cell antigen-2 (Sca-2), glycoprotein 38 (GP38) and cellular retinoic acid binding protein 1 (RABP1) [23]. These three TAAs are unaltered "self" antigens and, therefore, preexisting immunological tolerance to these antigens may exist in vivo.…”

Functional Studies of Lymphocytes Using RNAi Technology

“…siRNAs can be coupled with fusogenic peptides or linked to antibodies against cell surface receptor ligands in order to target specific cell types/tissues [3]. Co-immunization of animals with both DNA vaccines and siRNA has been successfully tested using the gene gun method targeting the epidermal DCs [31,32], Using a viral vector, such as the lentiviral vector mentioned above, may achieve persistent siRNA production and lengthen the duration of RNAi silencing [33][34][35][36]. Specifically targeting the immune system, particularly lymphocytes, in vivo is particularly challenging.…”