Antisense Expression Vector Research Articles

Suppression of invasion by inducible expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in B16-F10 melanoma cells.

We have previously shown that down-modulation (i.e., by antisense expression vector) of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a noninvasive, nontumorigenic cell line led to an acquisition of an invasive, tumorigenic, and metastatic ability in these cells. Our purpose was to examine whether increased levels of murine TIMP-1 can directly suppress the invasive ability of malignant cells. Murine B16-F10 melanoma cells were transfected with an expression vector to overproduce TIMP-1. Among these transfectants, we isolated five clonal cell lines (2-5, 2-8, 2-10, 6-5, and 6-9) that showed upregulation (i.e., overexpression) of TIMP-1. These cell lines had an increased basal level of TIMP-1 messenger RNA. TIMP-1 expression was under the control of the mouse metallothionein-I promoter, and four of these five clones (2-5, 2-8, 6-5, and 6-9) showed a threefold to 10-fold induction of TIMP-1 message when they were treated with 20 microM cadmium for 4 hours. An increase in TIMP-1 message led to an increase in TIMP-1 protein activity measured in the conditioned medium of clones 2-10 and 6-5. The invasive ability of the TIMP-1-upregulated cells was tested in a matrigel transwell invasion assay. All of the upregulated clones showed a significant reduction in their invasive ability, relative to the invasive ability of parental B16-F10 and the control 1-2 cell line; this reduction correlated with the level of TIMP-1 overexpression. Cadmium induction of TIMP-1 messenger RNA resulted in a further suppression of the invasive ability of the two inducible cell lines tested (i.e., 2-8 and 6-5). Our data demonstrate that a specific upregulation of murine TIMP-1 expression in B16-F10 melanoma cells directly suppresses their invasive ability.

Growth regulation of estrogen receptor-negative breast cancer cells transfected with complementary DNAs for estrogen receptor.

The growth of estrogen receptor (ER)-positive breast cancer cells is hormonally regulated, but the majority of breast cancers are ER negative and unresponsive to hormonal therapy. To test whether hormonal control over replication can be re-established in ER-negative cells, we transfected ER-negative MDA-MB-231 (clone 10A) cells with sense and antisense constitutive ER expression vectors containing the gene for either wild-type or mutant ER linked to the gene for neomycin resistance aminoglycoside phosphotransferase (neo). A Northern blot analysis was done on total RNA from eight of the 10 transfectant clones produced to detect messenger RNA coding for ER and neo, and a Western blot analysis was done on protein extracted from the cells of one mutant and two wild-type ER sense transfectant clones to determine the molecular weight of the ER in transfectants. Levels of ER in transfectants were measured both by enzyme immunoassay and by ligand-binding methods. To ascertain whether the ER in wild-type and mutant sense transfectants was functional, we tested the effects of 17 beta-estradiol (E2) and/or an antiestrogen, ICI 164,384, on 1) ER-activated gene regulation (by transient transfection of these cells a second time with a reporter plasmid containing an estrogen response element linked to the chloramphenicol acetyl transferase [CAT] gene), 2) induction of progesterone receptor, 3) DNA replication, and 4) cell cycle kinetics. Messenger RNA coding for ER and for neo was detectable in both sense and antisense transfectant clones. Sense transfectants (both mutant and wild-type) expressed ER protein with a molecular weight similar to that found in ER-positive control cells. By the ligand-binding method high levels of ER were detected in both wild-type and mutant transfectants, although by the enzyme immunoassay method lower levels were detected in mutant transfectants. ER from both wild-type and mutant sense transfectants appeared functional, since E2 stimulated the expression of reporter-linked CAT and of progesterone receptor in these transfectants. E2 inhibited DNA replication in wild-type sense transfectants at a concentration of 10(-10) M and mutant sense transfectants at a concentration of 10(-8) M, and ICI 164,384 blocked this effect. ER-negative breast cancer cells stably transfected with either a mutant or wild-type ER gene regain hormonal responsiveness; however, E2 inhibits rather than stimulates cell growth. Reactivation of quiescent ER may provide a novel therapeutic approach for controlling ER-negative breast cancers.

The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein.

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.

TGF-beta 1 is an autocrine-negative growth regulator of human colon carcinoma FET cells in vivo as revealed by transfection of an antisense expression vector.

Transforming growth factor-beta 1 (TGF-beta 1) has previously been implicated as a potential negative autocrine or paracrine growth regulator of certain cell types (Arteaga, C. L., R. J. Coffey, Jr., T. C. Dugger, C. M. McCutchen, H. L. Moses, and R. M. Lyons. 1990. Cell Growth & Differ. 1:367-374; Hafez, M. M., D. Infante, S. Winawer, and E. Friedman. 1990. Cell Growth & Differ. 1:617-626; Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Cell Regulation. 1:87-97). This is based mainly on experiments assessing the effects of exogenous TGF-beta 1 or neutralizing antibodies to TGF-beta 1 on normal or tumor cell proliferation in vitro. However, direct evidence demonstrating such a negative regulation of tumor cell growth in vivo is still lacking. To overcome this problem we have constructed and used an antisense expression vector for TGF-beta 1 as a means of regulating endogenous TGF-beta 1 expression in tumor cells. Antisense-transfected FET human colon carcinoma cells showed a fivefold reduction in TGF-beta 1 mRNA and 15-fold reduction in TGF-beta 1 secretion. Antisense mRNA was detected in transfected cells by an RNase protection assay. Compared to control cells, cultured antisense-transfected cells showed a reduction in lag phase time rather than a change in doubling time. Cloning efficiencies of transfected cells were four times greater than control cells in anchorage-independent assays. Control cells did not form tumors at 5 x 10(5) in athymic nude mice. Antisense-transfected cells formed tumors in 40% of animals injected. At higher inocula (1 x 10(6) cells) antisense-transfected cells formed tumors in 100% of animals injected, but control cells still failed to form tumors. These results show that TGF-beta 1 acts as a negative growth regulator of human colon carcinoma cells in vivo as well as in vitro. Acquisition of partial or full resistance to such inhibitory effects may therefore contribute to tumor development and progression.

The CCAAT/enhancer binding protein and its role in adipocyte differentiation: evidence for direct involvement in terminal adipocyte development.

During the course of differentiation of preadipocytes into adipocytes, several differentiation-linked genes are activated synchronously with morphological changes. To follow this process we have used 3T3-F442A cells, known to undergo adipocyte conversion with high frequency. Accumulation of lipid droplets in the cytoplasm constitutes an easily visualized sign of the terminally differentiated phenotype. In this report we demonstrate that expression of the CCAAT/enhancer binding protein (C/EBP) is an important factor in determining the ability to accumulate lipid droplets in terminally differentiated adipocytes. In one experiment we can suppress C/EBP expression through administration of hydrocortisone to differentiating 3T3-F442A cells, which is accompanied by an inability of the cells to accumulate lipid. In another experiment a C/EBP antisense expression vector has been stably introduced into 3T3-F442A cells and as compared with control cells, a 62% decrease of C/EBP mRNA (p less than 0.01) is demonstrated. This decrease of C/EBP mRNA is accompanied by a change in cellular morphology characterized by a reduced ability to form lipid droplets. We can also demonstrate a correlation between the degree of reduction of C/EBP mRNA and the amount of lipid present in the cells. These findings strongly support the view that C/EBP is a necessary component of terminal adipocyte differentiation.

Trial of Antisense RNA Inhibition of HIV Replication and Gene Expression

We examined the feasibility of antisense RNA inhibition of human immunodeficiency virus (HIV) replication. In the first experiment, we established CD4+ T-cell lines constitutively expressing various antisense HIV sequences using the retrovirus-mediated gene transfer technique. These cell lines were tested for their ability to withstand HIV de novo infection. In this challenge assay, however, we could not detect any significant difference in the survival rate between these genetically engineered cell lines and control T cells. In the second approach, the effects of antisense sequences on Tat expression were studied by monitoring the activities of reporter enzymes. A functional Tat expression vector and the antisense sequence expression vector were co-introduced into HeLa cells stably transfected with either the HIV-long terminal repeat (LTR) directed chloramphenicol acetyltransferase (CAT) or luciferase. Although the concentration of the antisense RNA was at least 10-fold higher than that of the sense Tat mRNA in cells, these antisense sequences could not inhibit transactivation of HIV-LTR. Regulation of HIV gene expression has proven to be very complicated and Tat transactivation of the HIV-LTR is extraordinarily strong. Consequently, it may be difficult to block HIV replication by the antisense strategy.

Antisense-mediated reduction in thrombospondin reverses the malignant phenotype of a human squamous carcinoma.

Thrombospondin (TSP) is a trimeric glycoprotein which is synthesized and incorporated into the extracellular matrix by a wide variety of cells. TSP is involved in a number of cellular processes which govern tumor cell behavior including mitogenesis, attachment, migration, and differentiation. To directly assess the role of TSP in tumor cell growth and spread, a human squamous carcinoma cell line, with high TSP production and an invasive phenotype, was transfected with a TSP cDNA antisense expression vector. Five unique transfected clones were obtained with reduced TSP production. Expression of the transfected antisense sequence in these clones was verified by a ribonuclease protection assay. These clones demonstrated reduced growth rates in vitro when compared with a vector transfected control. After subcutaneous inoculation into athymic mice, the antisense clones formed either no tumors or tumors that were slow growing and highly differentiated. This contrasted with the vector-transfected clone which produced poorly differentiated, rapidly growing, invasive tumors. Our results argue in favor of a direct role for TSP in determining the malignant phenotype of certain human tumors.

Involvement of retinoic acid nuclear receptors in retinoic acid-induced tissue transglutaminase gene expression in rat tracheal 2C5 cells

The involvement of retinoic acid nuclear receptors (RARs) in the induction of tissue transglutaminase (TG) by retinoic acid in rat tracheal 2C5 cells was determined. The levels of RAR alpha and RAR beta were altered in 2C5 cells by transfection with RAR expression vectors. Increased expression of RAR alpha increased the induction of tissue TG by retinoic acid. In contrast, decreased RAR alpha expression, using an antisense RAR alpha expression vector, diminished the normal level of tissue TG induction caused by retinoic acid. Transfectants overexpressing RAR beta were also more responsive to retinoic acid for the induction of tissue TG, although the magnitude of TG induction was not as great as resulted from RAR alpha overexpression. These results indicate that the levels of the RAR alpha and RAR beta dictate the magnitude of tissue TG induction by retinoic acid.

Expression of gene rrg is associated with reversion of NIH 3T3 transformed by LTR-c-H-ras.

A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha/beta. Persistent revertants stably transfected with rrg complementary DNA antisense expression vectors appeared transformed, had decreased amounts of rrg messenger RNA, and were tumorigenic in nude mice. Stable transfection with sense constructs did not alter the normal morphology, message level, or nontumorigenicity of the persistent revertant cell line.

Antisense RNA inhibition of HPRT synthesis

The abundant production of antisense hypoxanthine phosphoribosyltransferase (HPRT) RNA in NIH-3T3, COS, or HeLa cells leads to an inhibition of HPRT synthesis. HPRT enzyme levels in cells transfected with mouse HPRT antisense RNA expression vectors are reduced to less than 1% of parental cell activity, resulting in resistance to 6-thioguanine (6TG). The expression of antisense HPRT RNA leads to a marked reduction in the steady-state levels of endogenous HPRT mRNA. Furthermore, we demonstrate that intron-specific antisense RNA, complementary to sequences adjacent to splice donor or acceptor sites of the first intron of the mouse HPRT gene, are effective in depressing endogenous HPRT levels. These studies suggest that antisense RNA can inhibit gene expression in the nucleus, possibly by perturbing nuclear RNA processing.

Autoinduction of transforming growth factor beta 1 is mediated by the AP-1 complex.

The multifunctional actions of transforming growth factor beta 1 (TGF-beta 1) indicate that it has a pivotal control function in many physiological and pathological processes. An important property of TGF-beta 1 is its ability to activate its own mRNA expression and thereby increase its own secretion. Two distinct regions of the promoter of the TGF-beta 1 gene are responsive to autoregulation: one 5' to the upstream transcriptional start site and another located between the two major start sites. In both promoter regions, autoinduction is mediated by binding of the AP-1 (Jun-Fos) complex. An important contribution to this positive regulation is the autoactivation of c-jun transcription by AP-1. Cotransfection of antisense c-jun or antisense c-fos expression vectors prevents TGF-beta 1 autoinduction. These results demonstrate that both components of the AP-1 complex are required for TGF-beta 1 autoinduction. Induction of jun expression by TGF-beta 1, as well as jun autoinduction, may amplify the action of TGF-beta 1 during normal development and oncogenesis.

Autoinduction of Transforming Growth Factor β1 Is Mediated by the AP-1 Complex

The multifunctional actions of transforming growth factor beta 1 (TGF-beta 1) indicate that it has a pivotal control function in many physiological and pathological processes. An important property of TGF-beta 1 is its ability to activate its own mRNA expression and thereby increase its own secretion. Two distinct regions of the promoter of the TGF-beta 1 gene are responsive to autoregulation: one 5' to the upstream transcriptional start site and another located between the two major start sites. In both promoter regions, autoinduction is mediated by binding of the AP-1 (Jun-Fos) complex. An important contribution to this positive regulation is the autoactivation of c-jun transcription by AP-1. Cotransfection of antisense c-jun or antisense c-fos expression vectors prevents TGF-beta 1 autoinduction. These results demonstrate that both components of the AP-1 complex are required for TGF-beta 1 autoinduction. Induction of jun expression by TGF-beta 1, as well as jun autoinduction, may amplify the action of TGF-beta 1 during normal development and oncogenesis.

Reduction of EGF receptor levels in human tumor cells transfected with an antisense RNA expression vector

An expression vector was constructed from part of pSV2neo with the 3'-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction in cell-surface EGF receptor in response to ZnSO4. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

Functional consequences of anti-sense RNA-mediated inhibition of CD8 surface expression in a human T cell clone.

An experimental approach for defining the function of CD8 has been developed by linking anti-sense RNA mutagenesis and T cell cloning technologies. We have transfected an anti-sense CD8 episomal expression vector into a CD8+ nontransformed human T cell clone that is specific for the human class I alloantigen HLA-B35. Expression of CD8 on this T cell clone, JH.ARL.1, was selectively and efficiently inhibited. Stimulation of this CD8- variant with specific alloantigen resulted in a marked loss of a number of functional responses, including cytotoxicity, proliferation, IL-2 secretion, and IL-2-R expression. However, these same functional responses could be elicited with stimuli that do not require antigen recognition to activate the T cell (anti-CD3 mAbs, PHA). The results of our study support the hypothesis that CD8 is required for recognition of class I MHC alloantigens that results in activation of T cell functional responses.