Antisense Expression Vector Research Articles

Antisense Human Telomerase RNA Mediate d Inhibition of Telomere and Telomerase Activity in Human Gastric Cancer Cells

Objectivc To observe effects of antisense human telomerase RNA(hTR) on telomeric length (TRF) and telomerease activity of human gastric cancer cell SGC7901.Methods SGC7901 cell line was transfected with antisense hTR expression vector(pBBS-hTR) by lipofectamine,and expressions of hTR,telomere and telomerase activity in the gene transfected cells were detected by hybridization of nucleic acids and a telomeric repeat amplification protocol(TRAP).Results Clones containing antisense or control plasmids were selected 3 weeks after transfection by HyR selection and either antisense hTR expression had been enhanced or sense hTR expression had been inhibited 72h after transfection by specific stained RNA probe.Telomerase activity and mean TRF length were general low in the gene transfected cells in comparision with those in the control plasmid transfected cells.Conclusion Antisense hTR may either control TRF length by blocking the template region of hTR or serve as a effective target for treating gastric cancer by inhibiting telomerase.

Stearoyl-CoA Desaturase 1 Coding Sequences and Antisense RNA Affect Lipid Secretion in Transfected Chicken LMH Hepatoma Cells

Hepatic stearoyl CoA desaturase (SCD) activity in chickens from a fat line is higher than that of chickens from a lean line and correlates with plasma triacylglycerol concentrations. Furthermore, in these lines, the hepatic SCD1 mRNA level is positively correlated with the adipose tissue weight. To analyze the contribution of the SCD1 gene in the regulation of adiposity in the early stages of triacylglycerol secretion, SCD1 coding sequence and antisense RNA expression vectors were transfected in LMH cells. After selection, these cells were analyzed with regard to SCD1 expression and lipid secretion. The amounts of secreted triacylglycerols and phospholipids were shown to be higher in LMH cells transfected with the SCD1 gene, but reduced in those transfected with the SCD1 antisense sequences when compared to cells transfected with the vector alone (without SCD1 sequences). These results provide direct evidence that the expression of the SCD1 gene plays a major role in the triacylglycerol and phospholipid secretion process.

Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cell invasion by activating matrix metalloproteinase genes

Hepatocyte growth factor (HGF) is thought to play a role in cell motility and invasion. Matrix metalloproteinases (MMPs) have been implicated in invasion and metastasis of tumor cells. We have previously reported that the Ets-oncogene family transcription factor E1AF positively regulates transcription of MMP genes in transient expression assays and that overexpression of the E1AF gene confers an invasive phenotype on breast cancer cells. Here we examined the effect of HGF on E1AF and MMP gene expression in terms of the invasive potential of the oral squamous cell carcinoma cell line HSC3. HGF stimulated expression of the E1AF gene. The levels of MMP-1, -3 and -9 mRNAs increased in cells treated with HGF and correlated with E1AF upregulation. In contrast, no obvious upregulation of MMP-1 and -9 mRNA was observed in ASE1AFHSC3 cells transfected with the antisense E1AF expression vector into parental HSC3 cells. The wild-type MMP-9 gene promoter was activated by endogenous E1AF in HSC3 cells, and chloramphenicol acetyltransferase (CAT) activities increased when HGF was added to transfected cells. On the other hand, CAT activity was reduced to almost two-thirds of the wild-type activity when HSC3 cells were transfected with a CAT reporter plasmid driven by a mutant MMP-9 promoter lacking the Ets-binding site, and induction of CAT activity was not observed upon addition of HGF. Analysis of organotypic raft cultures revealed that HSC3 cells invaded and degraded collagen gel actively upon addition of HGF. These results suggest that HGF induces expression of the Ets-related E1AF transcription factor gene whose product in turn activates MMP genes and leads to oral cancer cell invasion.

DNA methyltransferase levels and altered CpG methylation in the total genome and in the GSTP1 gene in human glioma cells transfected with sense and antisense DNA methyltransferase cDNA

This study examines the efficacy of using plasmid expression vectors containing sense and antisense DNA MTase cDNA to both up- and downregulate intracellular DNA MTase levels in human glioma cells. The effects of the changes in MTase levels on global genomic DNA methylation and on the methylation status of CpG dinucleotides in the GSTP1 gene were determined in a glioma cell line that overexpresses the GSTP1 gene. In cells transfected with sense DNA MTase cDNA, MTase gene transcripts increased to a maximum of 2.5-fold at 24 h, while MTase activity increased to a maximum of 3.6-fold at 48 h. The effects of antisense MTase cDNA transfections were less pronounced, and levels of MTase gene transcripts and enzyme activity in transfectants were decreased to only, approximately, one-half the levels of controls. The alterations in DNA MTase expression were associated with corresponding changes in the level of global DNA methylation and in the methylation of the GSTP1 gene in the cells, however, with no detectable morphological or cytotoxic effects on the cells. No significant changes in GSTP1 gene expression were detected after the transfections, presumably because of the high levels of basal GSTP1 expression in the cells. Consequently, the p16 gene, known to be repressed transcriptionally by DNA methylation, was examined for the functional effects of the altered MTase levels. The results showed a 2-fold decrease in p16 gene transcripts with the sense MTase transfectants, while in the MTase antisense-transfected cells p16 transcript levels increased by 30%. Together, these results demonstrate the feasibility of using both sense and antisense DNA MTase expression vectors to regulate DNA MTase levels in glioma cells and that, over relatively short periods of time, the alterations in MTase activities are not deleterious to the cells. The system provides a model with which the role of DNA methylation in critical genes and DNA sequences can be investigated in glioma cells. J. Cell. Biochem. 77:372–381, 2000. © 2000 Wiley-Liss, Inc.

DNA methyltransferase levels and altered CpG methylation in the total genome and in the GSTP1 gene in human glioma cells transfected with sense and antisense DNA methyltransferase cDNA

This study examines the efficacy of using plasmid expression vectors containing sense and antisense DNA MTase cDNA to both up- and downregulate intracellular DNA MTase levels in human glioma cells. The effects of the changes in MTase levels on global genomic DNA methylation and on the methylation status of CpG dinucleotides in the GSTP1 gene were determined in a glioma cell line that overexpresses the GSTP1 gene. In cells transfected with sense DNA MTase cDNA, MTase gene transcripts increased to a maximum of 2. 5-fold at 24 h, while MTase activity increased to a maximum of 3. 6-fold at 48 h. The effects of antisense MTase cDNA transfections were less pronounced, and levels of MTase gene transcripts and enzyme activity in transfectants were decreased to only, approximately, one-half the levels of controls. The alterations in DNA MTase expression were associated with corresponding changes in the level of global DNA methylation and in the methylation of the GSTP1 gene in the cells, however, with no detectable morphological or cytotoxic effects on the cells. No significant changes in GSTP1 gene expression were detected after the transfections, presumably because of the high levels of basal GSTP1 expression in the cells. Consequently, the p16 gene, known to be repressed transcriptionally by DNA methylation, was examined for the functional effects of the altered MTase levels. The results showed a 2-fold decrease in p16 gene transcripts with the sense MTase transfectants, while in the MTase antisense-transfected cells p16 transcript levels increased by 30%. Together, these results demonstrate the feasibility of using both sense and antisense DNA MTase expression vectors to regulate DNA MTase levels in glioma cells and that, over relatively short periods of time, the alterations in MTase activities are not deleterious to the cells. The system provides a model with which the role of DNA methylation in critical genes and DNA sequences can be investigated in glioma cells.

The antisense BCL-2 oligonucleotide and CDNA expressing vector induce apoptosis in BCL-2-positive human hepatoma cells

The antisense BCL-2 oligonucleotide and CDNA expressing vector induce apoptosis in BCL-2-positive human hepatoma cells

TGFalpha is required for full expression of the transformed growth phenotype of NIH 3T3 cells overexpressing ornithine decarboxylase.

Ornithine decarboxylase (ODC) overexpressed from a heterologous promoter drives the tumorigenic transformation of NIH 3T3 cells and provides a model to investigate the underlying molecular mechanisms. These transformed cells, designated NODC cells, exhibit elevated levels of epidermal growth factor receptor (EGFR) tyrosine kinase (Tyr-k) activity relative to control transfected cells and inhibition of EGFR Tyr-k activation suppresses the transformed growth phenotype of these cells. Thus, ODC-induced transformation of NIH 3T3 cells appears to be mediated, at least in part, by enhanced signaling through the EGFR pathway. Here we extend these studies by evaluating: (i) the effects on growth regulation of overexpressing ODC in EGFR-deficient NIH 3T3 cells; (ii) the potential role of TGFalpha in mediating the EGFR-dependent transformation of NIH 3T3 cells by ODC. Disruption of EGFR-TGFalpha interactions either by deleting EGFR, by treatment with anti-TGFalpha neutralizing antibody or by transfection with a TGFalpha antisense expression vector suppressed acquisition of the full transformed growth phenotype. Specifically, the loss of contact inhibition and the capacity for clonogenic growth appear more dependent on EGFR-TGFalpha interactions than anchorage-independent growth in ODC-overexpressing cells. ODC overexpression does not alter the amount, localization or secretion of TGFalpha. Thus, TGFalpha is not the ODC-responsive component of the EGFR signaling pathway but appears to be critically involved in development of the transformed phenotype of NODC cells.

Suppression of proline-directed protein kinase F(A) expression inhibits the growth of human chronic myeloid leukaemia cells.

Initial studies revealed that proline-directed protein kinase FA(PDPK FA) was overexpressed in various cancerous tissues relative to normal controls. However, the functional role of overexpressed PDPK FAin cancer remains to be established. In this report, we explore the potential role of PDPK FAin leukaemia cell growth by investigating the effects of partial inhibition of this kinase on the malignant phenotype of human chronic myeloid leukaemia cells (K562). Cloning of PDPK FAcDNA and its recombinant antisense expression vector and PDPK FA-specific antibody were successfully developed. Two stable antisense clones of K562 cells were subcloned which expressed 70% and 45% of PDPK FArespectively, compared with control-transfected clone in both immunoprecipitate activity assay and immunoblot analysis. In sharp contrast, these two antisense clones expressed no significant suppression of any other related PDPK family members, indicating the specificity of these two antisense clones. Moreover, these antisense clones proportionally and potentially exhibited cell growth retardation, poor clonogenic growth in soft agar and loss of serum independence. The results demonstrate that specific antisense suppression of PDPK FAis sufficient to interfere with the growth of K562 cells, indicating that PDPK FAis essential for human chronic myeloid leukaemia cell growth. © 2000 Cancer Research Campaign

Beta globin gene inhibition by antisense RNA transcripts.

Sickle cell disease is caused by a mutation in the beta globin gene leading to hemoglobin S (Hb S) production. Several approaches have been explored to prevent Hb S polymerization in red blood cells and the symptoms associated with this disorder. To this end we tested a mammalian expression vector carrying a human beta globin antisense cDNA (pZeobetaAS) fragment in a mouse erythroleukemia cell line expressing the human gamma and beta globin genes. We observed a relative reduction in beta globin mRNA levels compared with gamma mRNA levels in the presence of pZeobetaAS. Moreover, analysis at the protein level showed an average 76% decrease in beta chains and a 517% increase in gamma chain biosynthesis. The inhibitory effect of the antisense vector on globin expression was maintained long term in culture. The expression vector pZeobetaAS was also transfected into primary erythroid progenitors to test its effects on globin genes undergoing normal developmental switching during differentiation. We observed a relative reduction of beta globin mRNA levels compared with gamma mRNA levels. These results support a novel role for antisense cDNA expression vectors as an alternative gene therapy strategy to inhibit betas gene expression in sickle cell disease. Gene Therapy (2000) 7, 438-444.

PAI-1 gene expression is regionally induced in wounded epithelial cell monolayers and required for injury repair.

Induced expression of plasminogen activator inhibitor type-1 (PAI-1), a major negative regulator of pericellular plasmin generation, accompanies wound repair in vitro and in vivo. Since transcriptional control of the PAI-1 gene is superimposed on a growth state-dependent program of cell activation (Kutz et al., 1997, J Cell Physiol 170:8-18), it was important to define potentially functional relationships between PAI-1 synthesis and subpopulations of cells that emerge during the process of injury repair in T2 renal epithelial cells. Specific cohorts of migratory and proliferating cells induced in response to monolayer trauma were spatially as well as temporally distinct. Migrating cells did not divide in the initial 12 to 20 h postinjury. After 24 h, S-phase cells were generally restricted to a region 1 to 2 mm from, and parallel to, the wound edge. Proliferation of wound bed cells occurred subsequent to wound closure, whereas the distal contact-inhibited monolayer remained generally quiescent. Hydroxyurea blockade indicated, however, that proliferation (most likely of cells immediately behind the motile "tongue") was necessary for maintenance of cell-to-cell cohesiveness in the advancing front, although the ability to migrate was independent of proliferation. PAI-1 mRNA expression was rapidly up-regulated in response to wounding with inductive kinetics approximating that of serum-stimulated cultures. Differential harvesting of T2 cell subpopulations, based on proximity to the injury site, prior to Northern assessments of PAI-1 mRNA abundance indicated that PAI-1 transcripts were restricted to cells immediately bordering the wound or actively migrating and not expressed by cells in the distal contact-inhibited monolayer regions. Such cell location-specific distribution of PAI-1-producing cells was confirmed by immunocytochemistry. PAI-1 synthesis in cells that locomoted into the wound field continued until injury closure. Down-regulation of PAI-1 synthesis and matrix deposition in renal epithelial cells, stably transfected with a PAI-1 antisense expression vector, significantly impaired wound closure. Transfection of the wound repair-deficient R/A epithelial line with a sense PAI-1 expression construct restored both approximately normal levels of PAI-1 synthesis and repair ability. These data indicate that PAI-1 induction is an early event in creation of the wound-activated phenotype and appears to participate in the regulation of renal epithelial cell motility during in vitro injury resolution.

Disruption of transforming growth factor beta-regulated laminin receptor function by expression of antisense laminin, a chain RNA in human colon cancer cells.

Transforming growth factor beta1 (TGFbeta) simultaneously induces the expression of fibronectin, fibronectin receptor, laminin, and laminin receptor (alpha6beta1 integrin) in the human colon cancer cell line Moser (Int J Cancer, 57:742, 1994). Induction of fibronectin and induction of fibronectin receptor by TGFB are tightly coupled, and disrupting fibronectin induction disrupts the induction of fibronectin receptor and cellular adhesion to fibronectin (J Cellular Physiol, 170:138, 1997). We recently demonstrated the efficacy of using antisense chain-specific laminin RNA expression vectors to disrupt the induction by TGFP of the multichain laminin molecule (J Cellular Physiol, 178:296, 1999). We now show in this report that Moser cells used alpha6 and beta1 integrins to adhere to laminin, and, as is the fibronectin and fibronectin receptor system, disrupting the induction by TGFbeta of the ligand laminin by the expression of antisense laminin A chain RNA disrupted the induction of 125I-laminin binding and cellular adhesion to laminin. Disrupting laminin induction also blocked the induction of alpha6 and beta1 integrin laminin receptor by TGFbeta. We conclude that disrupting the induction of the ligand laminin by TGFbeta disrupts TGFbeta-regulated laminin receptor function by suppressing the induction of alpha6 and beta1 integrins. Therefore, targeted disruption of the ligand laminin may be an effective means in disrupting the function of both the ligand and its receptor in cells that utilize the laminin and laminin receptor system in malignant cell behavior.

Generation of a Ribozyme-Adenoviral Vector Against K-ras Mutant Human Lung Cancer Cells

ras mutations represent one of the most common oncogenetic lesions in human non-small cell lung cancer (NSCLC) and adversely affect the survival of patients afflicted with this disease. ras-directed gene therapy in the past employed primarily antisense oligonucleotides (AS-ODN) or expression vectors (such as a viral vector construct) that deliver the antisense sequence to inactivate the mutant oncogene message. These approaches produced minimal toxicity, and yet were limited in efficacy. Ribozymes present a viable alternative in antisense therapy by virtue of their renewable catalytic capability for site-specific RNA cleavage. We recently produced an adenoviral vector with a hammerhead ribozyme transgene (KRbz) that is specific for the K-ras codon 12 mutant sequence GUU, given the considerations that (a) in the United States, approx 30% of human NSCLCs express K-ras oncogene mutations, nearly all of which reside in codon 12; (b) anti-K-ras, anti-H, as well as anti-N-ras hammerhead ribozymes are potent growth inhibitors in various human cancers tested; and (c) in vitro and animal model studies suggest that ribozymes directed at oncogene (K- and H-ras C-fos, BCR-ABL) or human immunodeficiency viral gene messages are more effective than their antisense counterpart. This article describes the techniques involved in the production of the KRbz-adenoviral vector that is specific for the K-ras mutation GTT, and summarizes its in vivo antitumor effect against NSCLC xenografts expressing the relevant K-ras mutation in athymic mice.

PEG-3, a nontransforming cancer progression gene, is a positive regulator of cancer aggressiveness and angiogenesis.

Cancer is a progressive disease culminating in acquisition of metastatic potential by a subset of evolving tumor cells. Generation of an adequate blood supply in tumors by production of new blood vessels, angiogenesis, is a defining element in this process. Although extensively investigated, the precise molecular events underlying tumor development, cancer progression, and angiogenesis remain unclear. Subtraction hybridization identified a genetic element, progression elevated gene-3 (PEG-3), whose expression directly correlates with cancer progression and acquisition of oncogenic potential by transformed rodent cells. We presently demonstrate that forced expression of PEG-3 in tumorigenic rodent cells, and in human cancer cells, increases their oncogenic potential in nude mice as reflected by a shorter tumor latency time and the production of larger tumors with increased vascularization. Moreover, inhibiting endogenous PEG-3 expression in progressed rodent cancer cells by stable expression of an antisense expression vector extinguishes the progressed cancer phenotype. Cancer aggressiveness of PEG-3 expressing rodent cells correlates directly with increased RNA transcription, elevated mRNA levels, and augmented secretion of vascular endothelial growth factor (VEGF). Furthermore, transient ectopic expression of PEG-3 transcriptionally activates VEGF in transformed rodent and human cancer cells. Taken together these data demonstrate that PEG-3 is a positive regulator of cancer aggressiveness, a process regulated by augmented VEGF production. These studies also support an association between expression of a single nontransforming cancer progression-inducing gene, PEG-3, and the processes of cancer aggressiveness and angiogenesis. In these contexts, PEG-3 may represent an important target molecule for developing cancer therapeutics and inhibitors of angiogenesis.

Functional antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) trans-factors for the regulation of fatty acid synthase promoter in adipocytes

We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA. Using the FAS gene as a model of an ADD1-regulated promoter in transiently transfected isolated rat adipocytes or mature 3T3-L1 adipocytes, a potent inhibition of the activity of the FAS-chloramphenicol acetyltransferase reporter gene was observed by overexpression of Id2 or Id3. Reciprocally, co-transfection of Id3 antisense and ADD1 expression vectors in preadipocytes potentiated the ADD1/SREBP-1c effect on the FAS promoter activity. Finally, in the non adipogenic NIH-3T3 cell line, most of the ADD1-mediated trans-activation of the FAS promoter was counteracted by co-transfection of Id2 or Id3 expression vectors. Previous studies have indicated Id gene expression to be down-regulated during adipogenesis [Moldes, Lasnier, Fève, Pairault and Djian (1997) Mol. Cell. Biol. 17, 1796-1804]. We here demonstrated that there was a dramatic rise of Id2 and Id3 mRNA levels when 3T3-L1 adipocytes or isolated rat fat cells were exposed to lipolytic and anti-lipogenic agents, forskolin and isoproterenol. Taken together, our data show that Id products are functionally involved in modulating ADD1/SREBP-1c transcriptional activity, and thus lipogenesis in adipocytes.

Possible involvement of thioredoxin reductase as well as thioredoxin in cellular sensitivity to cis-diamminedichloroplatinum (II)

The thioredoxin (TRX) system, composed of nicotinamide adenine dinucleotide phosphate (reduced form), TRX, and TRX reductase (TRXR), has multiple biologic functions via thiol-mediated redox control. In this study, we investigated the relationship between intracellular TRXR levels and cellular sensitivity to cis-diamminedichloroplatinum (II) (CDDP). HeLa, a human cervical carcinoma cell line, cultured with CDDP showed a time- and dose-dependent reduction of intracellular TRXR activity, which was well correlated with the decrease in cell viability after exposure to CDDP. In a cell-free system, CDDP was found to directly inactivate the reduced form of purified human TRXR. The CDDP-resistant variants of HeLa cells, established by continuous exposure to CDDP, exhibited an increased expression and activity of TRXR as well as TRX compared with the parental cells. In addition, sodium selenate, an inhibitor of TRXR, was found to increase the susceptibility to CDDP in the CDDP-resistant cells. Moreover, the HeLa cells transfected with an antisense TRXR RNA expression vector to reduce the intracellular enzyme activity displayed an enhanced sensitivity to CDDP. Taken together with previous reports on TRX, these results indicate the possible involvement of TRXR as well as TRX in the cellular sensitivity and resistance to CDDP.

Retrovirus producer cells encoding antisense VEGF prolong survival of rats with intracranial GS9L gliomas

With increasing size tumors are continually dependent on a functional blood vessel system to guarantee the supply with oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key mediator not only of developmental but also of hypoxia-mediated and tumor-induced angiogenesis. Gene therapy using antisense VEGF with the aim to inhibit tumor angiogenesis may be a successful strategy for the treatment of highly vascular and invasive malignant gliomas. We investigated whether retrovirus producer cells encoding antisense VEGF can be used for in vivo gene transfer. The full length mouse VEGF164 cDNA was cloned in a sense and antisense direction into the retroviral expression vector pLEN. pLEN–VEGF (sense) and pLEN–FGEV (antisense) expression vectors were used to transfect the packaging cell line GP+E86 and to establish ecotropic virus producer cell lines. GP+E86:LEN–FGEV (#5) cells showed high expression of antisense VEGF mRNA, whereas GP+E86:LEN–VEGF (#8) showed high expression of sense VEGF mRNA and active VEGF protein. Co-implantation of GS-9L cells with retrovirus producing cells containing the antisense VEGF construct into the brains of syngeneic rats showed a statistically significant inhibition of tumor growth and prolongation of survival time, while co-implantation of retrovirus producer cells containing the sense VEGF expression vector resulted in an increasing tumor growth and reduced survival time of the rats compared to control animals. Histological analysis of the tumors co-implanted with GP+E86:LEN–FGEV (#5) cells showed the suppression of angiogenesis, high degree of necrosis and no evidence of a significant immune response. Expression of antisense VEGF mRNA in these tumors was confirmed by in situ hybridization analysis. This is the first report demonstrating the potential utility of virus producer cells as in vivo gene transfer vehicles for antisense VEGF gene therapy of malignant gliomas.

CREB mediates the cAMP-responsiveness of the tyrosine hydroxylase gene: use of an antisense RNA strategy to produce CREB-deficient PC12 cell lines

cAMP initiates the PKA signaling cascade in rat pheochromocytoma PC12 cells, resulting in transcriptional activation of the tyrosine hydroxylase (TH) gene. This effect is mediated primarily through the cAMP responsive element (CRE), located at position −45 to −38 within the TH gene promoter. In this study, we applied an antisense RNA strategy to evaluate the role of the cAMP responsive element binding protein (CREB) in regulating TH gene expression. CREB antisense RNA expression vectors were stably introduced into PC12 cells to generate cell lines deficient in CREB. CREB protein and mRNA levels were diminished up to 90% in the stably transfected cell lines. Promoter analysis experiments demonstrated that cAMP-mediated inducibility of either TH gene proximal promoter activity or the activity of the TH CRE by itself fused upstream of a basal promoter was diminished in CREB-deficient cell lines. PKA activity in the CREB-deficient cell lines was comparable to the activity in control cell lines. In addition, neither ATF1, nor CREM proteins were significantly down-regulated in the CREB-deficient cells. Most significantly, the cAMP-inducibility of endogenous TH mRNA was completely blocked in the CREB-deficient cells, indicating that the response of the endogenous gene to cAMP was dependent on CREB. These results support the hypothesis that CREB (not other CRE-binding proteins) is the key transcription factor that is required for regulating TH gene expression in response to cAMP. Furthermore, our studies indicate that these CREB-deficient PC12 cells are excellent tools to study the participation of CREB in gene regulation.

Inhibition of Oncogene Expression Using Vector-Generated RNA Antisense

Antisense RNA expression vectors have been developed relatively recently as a means to study the role of specific oncogenes in malignant transformation. In this paper, strategies for the construction of antisense plasmid vectors from commercially available reagents are described. Techniques for the introduction of these vectors into cell lines and tumors are also described and preferred methods for the evaluation of biological effects are presented. Lastly, using specific examples, the limitations and potential artifacts associated with antisense vector use in the study of tumorigenesis are discussed.

Alteration of ganglioside composition by stable transfection with antisense vectors against GD3-synthase gene expression.

Gangliosides are ubiquitous components of mammalian cells. Their expression is frequently altered in many tumor types. We previously showed that alteration of the ganglioside composition often resulted in changes in cellular morphology and differentiation of cultured cells. In this study, we targeted sialyltransferase gene expression by the antisense knockdown experiment, and the results showed that inhibition of the expression of gangliosides GD3 and O-acetylated GD3 (OAc-GD3) in the neuroblastoma F-11 cells greatly reduced the tumor growth in nude mice. The sense and antisense vectors containing either a 5' end fragment or the entire sequence of the cDNA coding for GD3-synthase were prepared and used in separate experiments to transfect the F-11 cells which express high levels of gangliosides GD3 and OAc-GD3. Single clones were isolated and expanded. Both the activity of the GD3-synthase and the concentrations of GD3 and OAc-GD3 in the antisense-transfected cells were dramatically decreased as a result of transfection with the antisense expression vectors. Further characterization of the antisense-transfected cells showed reduced rates of cell growth and neurite formation and changes in cellular morphology. When the cells were inoculated in athymic nude mice, the tumor growth rate was remarkably suppressed although the tumor incidence was not affected by the altered ganglioside composition. These results indicate that the tumor-associated ganglioside(s) is(are) involved in regulation of tumor growth, probably through the stimulation of angiogenesis of the tumor.

Inhibition of metastasis to lung of a human nasopharyngeal carcinoma cell line CNE- 2L2 transfected with pRc/ CMV-antisense 6A8 cDNA in nude mice

The growth of CNE-2L2 cell, a cloned line of human nasopharyngeal carcinoma with a high potentiality of metastasis to lung was inhibited to a certain extent after transfection with a recombinant antisense expression vector of a cDNA encoding a human alpha-mannosidase (pRc/CMV-antisense 6A8 cDNA) (the Genbank accession number of 6A8 cDNA is U37248) in comparison with that of the cell transfected with the Mock and of the wild cell. Two months after a subcutaneous inoculation of CNE-2L2 cell into the axilla of nude mice metastatic lesions in the lung were observed in 9/10 mice (90%) with grade III in 8 mice and grade II in one mouse in the wild cell group, in 6/8 mice (75 %) with grade III in one mouse, grade II in 2 mice and grade I in 3 mice in the Mock-transfection group, in only 3/10 mice (30%) with all grade I in pRc/CMV-antisense 6A8 cDNA-transfection group.