Sense Oligonucleotides Research Articles

Inhibition of smooth muscle cell proliferation and migration in vitro by antisense oligonucleotide to c-myb

Smooth muscle cell (SMC) migration and proliferation are prominent features of intimal hyperplasia. Previous studies have shown that inhibition of c-myb inhibits arterial SMC proliferation. Our goal was to evaluate the effect of an antisense oligonucleotide targeted to c-myb on the proliferation and migration of SMC explanted from synthetic vascular grafts. SMCs were enzymatically removed from aortas and Dacron grafts explanted from dogs (n = 5). For proliferation studies, quiescent SMCs were incubated with either 0.0, 0.5, 5.0, or 10.0 microM antisense (GTGTCGGGGTCTCCGGGC) or sense (GCCCGGAGACCCCGACAC) oligonucleotides to c-myb. Proliferation was measured after 24 hours by incorporation of [3H]thymidine. Migration was assessed 24 hours after a razor injury. Antisense to c-myb consistently inhibited proliferation and migration of both native aortic and graft SMCs in a dose-dependent fashion. At a concentration of 10 microM antisense oligonucleotide, aortic and graft SMC proliferation rates were 32% +/- 20% and 56% +/- 9% of control samples, respectively. At 25 microM antisense, the number of migrating aortic and graft SMCs decreased to 41.9% +/- 26.8% and 51.9% +/- 34.1% of control samples, respectively. Our results suggest that antisense oligonucleotides to c-myb may be useful in the inhibition of SMC proliferation and migration associated with development of intimal hyperplasia.

Repression of Transforming Growth Factor β1 Protein by Antisense Oligonucleotide-induced Increase of Adrenal Cell Differentiated Functions

Transforming growth factor beta 1 (TGF beta 1) is a potent inhibitor of several differentiated functions in bovine adrenal fasciculata cells (BAC). In addition, these cells express and secrete this factor. To determine whether this peptide plays an autocrine role in BAC, cells were transfected with 10 microM unmodified sense (SON) or antisense (AON) oligonucleotide complementary to the translation initiation region of the TGF beta 1 mRNA in an attempt to inhibit TGF beta 1 protein synthesis. We investigated first, the cellular uptake, the stability, and the intracellular distribution of 32P-labeled TGF beta 1 AON and SON; and second, the effects of both oligonucleotides on BAC specific functions. We have demonstrated that in BAC, the TGF beta 1 AON uptake reached a plateau after 8 h of transfection (16% of the radioactivity added) and remained fairly constant for at least 24 h. In contrast, the uptake of TGF beta 1 SON reached a plateau after 2 h of transfection (8% of the radioactivity added), remained stable for only 3 h, and then declined. After 8 h of transfection, followed by 44 h of culture without oligonucleotides, the intracellular level of TGF beta 1 AON was still high with about 8% of the radioactivity added, whereas that of TGF beta 1 SON represented only 1.2%. Moreover, AON was present in the cytoplasmic and nuclear fractions, and it was hybridized in both compartments. However, TGF beta 1 SON was present mainly in the cytoplasmic fraction where it was not hybridized. Neither TGF beta 1 AON nor SON modified TGF beta 1 mRNA levels; however, TGF beta 1 AON, but not SON, caused the disappearance of TGF beta 1 immunoreactivity inside the cells. Finally, the steroidogenic responsiveness of BAC transfected with TGF beta 1 AON increased about 2-fold, and this was associated with a 2-fold increase of the mRNA levels of both cytochrome P450 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase. Neither TGF beta 1 SON nor a scrambled oligonucleotide containing the same number of G nucleotides as TGF beta 1 AON had any effect on these parameters. Thus, these studies demonstrate that TGF beta 1 has an autocrine inhibitory effect on BAC differentiated functions, an effect that can be overcome by TGF beta 1 AON.

Antisense oligonucleotide to the 70-kDa heat shock cognate protein inhibits synthesis of myelin basic protein.

Transfection of rat oligodendrocytes with an oligonucleotide sequence complementary to the mRNA encoding the initial ten amino acids of the rat 70-kDa heat shock cognate protein (HSC70) resulted in a rapid (within 24 h) and significant reduction in HSC70 synthesis (69% of control cells transfected with sense oligonucleotide). A further decrease to approximately 44% of controls was detected after 2 days. At that time, HSC70 protein content fell to approximately 49% of controls, and a significant reduction in the synthesis of myelin basic protein (MBP) was first detected (66% of controls). After 5 days, HSC70 synthesis returned to control levels. As HSC70 protein content recovered, so did the synthesis of MBP. Throughout the 5-day experimental period, only minor changes were detected in cell morphology, overall pattern of protein synthesis and the synthesis and content of proteolipid protein (PLP) and the pi isoenzyme of glutathione-S-transferase (pi). These data show that when HSC70 protein content is sufficiently reduced by antisense oligonucleotide, synthesis of MBP (but not PLP or pi) is correspondingly down-regulated, and provide evidence consistent with the role of HSC70 as a chaperone for MBP.

IMMORTALIZED FETAL RAT NEURONAL CELLS SYNTHESIZE NEUROPEPTIDE Y.† 384

Neuropeptide Y (NPY), an orexigenic peptide synthesized in-vivo within the arcuate nucleus and released in the paraventricular nucleus, causes hyperphagia and anxiolysis. To determine the presence of NPY synthesis and secretion by fetal rat neural cells that were immortalized by the temperature sensitive large T antigen, we undertook the present study. Using undifferentiated (33°C, serum-containing medium) and differentiated(39°C, serum-free defined medium) neural cells predominantly expressing neuronal (neurofilament [NF] positive, glial fibrillary acidic protein [GFAP] negative), and glial (NF -ve, GFAP +ve) phenotypes, we characterized the presence of NPY mRNA by Northern blot analysis (n=2), cellular peptide by immunohistochemical analysis (n=4), and extracellular secretion of the NPY peptide over 20 min by radioimmunoassay of Kreb's buffer conditioned medium(n=10). Differentiation of neuronal and glial enriched cell cultures led to a 24-fold and 13-fold increase in NPY mRNA (0.8 kb) concentrations respectively(p <0.01). A similar change was noted in the number of cells expressing NPY immunoreactivity, this immunoreactivity being noted only in well differentiated neuronal cells and their processes. The increase in NPY immunoreactive cell number with differentiation was not paralleled by an increase in the extracellularly secreted NPY peptide levels. Employing 10μm of antisense oligonucleotide complementary to 20 bp downstream starting from the ATG-translation start site codon of the rat preproNPY gene, the number of NPY immunoreactive neuronal cells diminished 6-fold when compared to corresponding sense and missense oligonucleotide treated cells (n=3 each; p.<0.05). We conclude that 1] immortalized fetal neuronal cells synthesize and actively secrete NPY, the rate of synthesis being higher in differentiated cells, and 2] appropriately synthesized antisense oligonucleotides suppress neuronal NPY peptide production. These in-vitro studies will set the stage for undertaking in-vivo selective neuronal knock out of the hypothalamic NPY, thereby allowing phenotypic characterization of postnatal animals in the absence of hypothalamic NPY production.

Non target-derived roles of the neurotrophins.

The hypothesis that target-derived neurotrophic factors are essential for the survival, differentiation and maintenance of sensory, sympathetic and motor neurons has been well supported by analysis of mice bearing null mutations in the neurotrophins and their receptors. However, the localization of brain-derived neurotrophic factor (BDNF) in a population of dorsal root ganglia (DRG) sensory neurons (Ernfors et al. 1990b; Ernfors & Persson 1991; Schecterson & Bothwell 1992) suggested the additional possibility that BDNF could act in a paracrine or autocrine manner to mediate neuronal survival. We tested this hypothesis in cultured adult DRG neurons, which survive as single cells in microwells in the absence of added trophic factors (Lindsay 1988). About 35% of these neurons were specifically killed by BDNF antisense oligonucleotide administration in a dose-dependent manner, with no effect of sense oligonucleotides. Antisense administration was accompanied by an 80% decrease in BDNF protein levels over the first 24 h of treatment (Acheson et al. 1995). The BDNF autocrine loop that we propose to be present in sensory neurons may be representative of a broader phenomenon in the nervous system as a whole, where the balance of neurotrophic support may shift during development from target-derived to paracrine or autocrine modes. Perhaps as a consequence of this developmental shift, the survival of both peripheral nervous system (PNS) and central nervous system (CNS) neurons in the adult is less affected by axotomy or target removal when compared to their response during development.

Facilitator oligonucleotides increase ribozyme RNA binding to full-length RNA substrates in vitro

Primer extension arrest (PEA) studies have demonstrated that antisense oligonucleotides (ß112C, ß114C), which lie upstream of a ribozyme targeted to ß-amyloid peptide precursor (ßAPP) mRNA, but not sense oligonucleotides (ß112S, ß116S) or a scrambled oligonucleotide, ß116M, affect ribozyme-mediated cleavage in vitro. Substrate dissociation experiments revealed that the ribozyme binding site in this mRNA was masked; PEA kinetics showed the association of the ribozyme and substrate was enhanced by antisense oligonucleotide binding. These studies suggest that masked ribozyme cleavage sites that may occur in disease-causing mRNAs can be targeted for degradation using “facilitator” oligonucleotides.

Expression of fibroblast growth factor-1 (FGF-1), FGF-2 and FGF receptor-1 in a human salivary-gland adenocarcinoma cell line: evidence of growth.

Fibroblast growth factor-1 (FGF-1) and FGF-2 are heparin-binding polype ptides which express potent mitogenic properties in neoplastic cells. In the present study, we have examined the contribution of endogenous FGF-1 and FGF-2 to the autocrine growth of HSY human salivary-gland adenocarcinoma cells in vitro. Using specific monoclonal antibodies against FGF-1 and FGF-2, immunohistochemical analysis of HSY cells revealed strong expression of both FGF-1 and FGF-2 in the cytoplasm and nucleus. Consistent with these data, 2 molecular mass species of FGF-1 (16 and 18 kDa) and 3 FGF-2 (18, 24 and 27 kDa) were identified in HSY cells by Western-blot analysis. Scatchard analysis of FGF binding sites on HSY cells indicated the presence of 23,000 [125I]FGF-1 binding sites/cells with a dissociation constant (KD) of 178 pM and 13,000 [125I]FGF-2 binding sites/cell with a KD of 102 pM. In addition, HSY cells were shown to express the mRNA for FGF receptor-1 (FGFR-1) by reverse transcription-polymerase chain reaction (RT-PCR), confirming the existence of high-affinity FGF binding sites. The influence of endogenous FGF-1 and FGF-2 on HSY cell growth was evaluated by suppressing the expression and activity of FGF by using anti-sense oligonucleotides and neutralizing antibodies. The addition of 50 micron FGF-1-specific anti-sense oligonucleotides to HSY cells resulted in a 61% inhibition of cell growth, while 50 microM FGF-2-specific anti-sense oligonucleotides resulted in a 76% inhibition. These effects were dose-dependent and specific, since sense oligonucleotides were ineffective in inhibiting HSY cell growth at the same concentration. Furthermore, HSY cell growth was suppressed in the presence of anti-FGF-1 or anti-FGF-2 neutralizing antibody, resulting in a 58% inhibition at 8 micromilligrams/ml. Our observations suggest that FGF-1 and FGF-2 may act as autocrine regulators by interacting with FGF receptors on HSY cells.

Evidence that Glyceraldehyde‐3‐Phosphate Dehydrogenase Is Involved in Age‐Induced Apoptosis in Mature Cerebellar Neurons in Culture

Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.

Transforming growth factor beta mediates the angiotensin-II-induced stimulation of collagen type IV synthesis in cultured murine proximal tubular cells

Angiotensin II (Ang II) stimulates synthesis of type IV collagen in a cultured murine proximal tubular cell line (MCT cells). In addition, Ang II also induces the expression of TGF-beta1 in these cells. Since TGF-beta has well-known stimulatory effects on the transcription of various collagens, we tested whether the Ang-II-mediated stimulation of type IV collagen is due to induction of endogenous TGF-beta1 synthesis in MCT cells. A neutralizing monoclonal anti-TGF-beta1-3 antibody abolished the Ang II-stimulated release of type IV collagen in culture supernatants. The anti-TGF-beta1-3 antibody also partly blocked Ang-II-mediated incorporation of 3[H]proline into de novo synthesized collagens. Moreover, 5 microM TGF-beta1 antisense oligonucleotides, but not the same concentration of sense oligonucleotides, completely blocked Ang-II-stimulated 3[H]proline incorporation. MCT cells incubated with TGF-beta1 antisense phosphorothioate-modified oligonucleotides failed to synthesize TGF-beta1 protein after Ang II treatment as measured by a sandwich ELISA in culture supernatants. SDS-polyacrylamide electrophoresis of 3[H]proline-labelled collagens and comparison with standard collagens also demonstrated that the neutralizing anti-TGF-1-3 antibody abolished the Ang-II-mediated stimulation in type IV collagen. Semiquantitative cDNA amplification for collagen type alpha1 (IV) transcripts revealed that the anti-TGF-beta1-3 antibody abrogates the increase in mRNA after Ang II treatment. Transient transfection studies in MCT cells using murine collagen alpha1 (IV) enhancer/promoter constructs also demonstrated the suppressive effect of the neutralizing antibody on Ang-II-stimulated gene transcription. Our data collectively suggest that the Ang-II-mediated increase in type IV collagen in MCT cells is mediated by endogenous synthesis and autocrine action of TGF-beta1. These findings may be important in changes of the tubulointerstitial architecture during the progression of renal disease.

Autocrine induction of DNA synthesis by mechanical injury of cultured smooth muscle cells. Potential role of FGF and PDGF.

To determine whether replication of arterial smooth muscle cells (SMCs) in response to mechanical injury would occur in the absence of serum and other cells, we created an in vitro model in which confluent, growth-arrested cultures of rat SMCs were injured by gentle pressure of a soft plastic tube and then kept in serum-free medium for up to 4 days. Replication of SMCs in and around the injury, as measured by tritiated thymidine incorporation, was noted within 24 hours and peaked at 48 hours after injury, whereas noninjured cells remained quiescent. An increased expression of platelet-derived growth factor (PDGF) A mRNA, noted 6 hours after injury, was followed by an increased PDGF AA immunoreactivity in SMCs in and around the zone of injury at 24 and 48 hours after injury. A PDGF A chain antisense oligonucleotide inhibited 87.0 +/- 4.0% (P < .005) of SMC replication in the injury zone, whereas the corresponding sense oligonucleotide reduced SMC replication by only 37.2%. An antibody to fibroblast growth factor (FGF) almost completely inhibited SMC replication in the injured zone, whereas an antibody to PDGF AA was without effect. Incubation of SMCs with FGF increased PDGF A mRNA levels in SMCs, and 5 mumol/L PDGF A antisense oligonucleotides reduced FGF-induced SMC replication by 62%. Taken together, these results demonstrate that injured rat SMCs in culture release FGF that activates DNA synthesis of neighboring SMCs both by a direct mechanism and by stimulating the production of PDGF AA.

Downregulation of Msx-2 expression results in chondrogenesis in the medial region of the avian mandible.

Homeobox-containing genes are thought to be regulators of pattern formation during vertebrate development. We have previously characterized regions in the developing chick mandible expressing either Msx-1 or Msx-2 in terms of their chondrogenic potential, rates of cell proliferation, and their correlation with regions of cell death. These experiments suggest that Msx-1 may be involved in outgrowth of the mandibular arch, and Msx-2 may be involved in delineating the non-chondrogenic region of the medial part of the mandibular arch. To further examine the possibility that expression of Msx-2 may be involved in preventing chondrogenesis in the medial region, mandibular arch explants from stage 23 chick embryos were cultured for 4 days in media in the absence of serum but in the presence of 20-30 microM Msx-2 sense or antisense oligonucleotides (18 mers). In explants grown in either control media or with the sense oligonucleotide two rods of cartilage separated by a cartilage free area located in the medial region of the mandible were formed. In explants treated with Msx-2 antisense oligonucleotide cartilage formation was observed in the medial region of the mandible resulting in the fusion of the two bilateral rods at the midline. These results suggest a negative relationship between Msx-2 expression and chondrogenesis in the medial region of the developing mandible.

Beta ig-h3 is synthesized by corneal epithelium and perhaps endotheliumin Fuchs' dystrophic corneas.

Deposition of abnormal sub-epithelial matrix and posterior collagenous layer by epithelium and endothelium, respectively, in Fuchs' dystrophy gives us the opportunity to determine if these tissues synthesize beta ig-h3. Immunohisto-/immunocytochemistry of corneas were conducted with rabbit anti-human beta ig-h3 and monoclonal anti-human type VI collagen. Labeled sense and anti-sense beta ig-h3 oligonucleotide probes were used for in situ hybridization. beta ig-h3-specific fluorescence was found just beneath detached epithelium in the sub-epithelial matrix, abnormal Descemet's membrane and posterior collagenous layer. Type VI collagen co-localized with beta ig-h3 within abnormal sub-epithelial matrix and corneal stroma adjacent to Descemet's membrane. beta ig-h3 mRNA was detected in corneal epithelium of dystrophic corneas. Expression of beta ig-h3 in sub-epithelial matrix and posterior collagenous layer of Fuchs' dystrophy is consistent with the synthesis of new extracellular matrices by epithelial and endothelial tissues. beta ig-h3 mRNA in corneal epithelium further supports an epithelial source of this protein. Endothelial synthesis of beta ig-h3 is based on circumstantial evidence due to cell loss during surgical and histological procedures. Co-localization of beta ig-h3 with type VI collagen in abnormal sub-epithelial matrix and at the stromal/Descemet's membrane interface suggest this collagen in association with beta ig-h3 interacts with these tissues and anchors them to the adjacent stroma.

Initiation of impaired outer segment degradation in vivo using an antisense oligonucleotide.

This paper describes the first successful in vivo application of antisense DNA technology to induce the accumulation of photoreceptor outer segment derived debris in the retina. An antisense oligonucleotide (CatSC), which was previously demonstrated to be an effective tool to induce debris accumulation in vitro, was injected into the vitreous of pigmented and non-pigmented rats. The animals were euthanased 7 days after the injections. The number of inclusions significantly increased in the RPE layer of Long Evans and RCS-rdy + rats injected with 66 ug of CatSC to 96.2 +/- 13.6 (SD) (p < 0.0003) and 204.2 +/- 39.3 (SD) (p < 0.0001), respectively. The difference between the number of phagosome-like inclusions present in control saline, 6.6 ug of CatSC or 66 ug of sense oligonucleotide (S1) injected animals was not statistically significant. There were no abnormalities observed in the inner layers of the retina but the accumulation of phagosome-like inclusions was accompanied by disorganisation in the apices of outer segments. The large number of inclusions found in CatSC treated animals showed the characteristics of phagosomes containing stacks of undigested photoreceptor outer segment membranes which suggest that the lysosomal digestion process was halted or at least slowed down by the antisense oligonucleotide.

Development of a Novel Scintillation Proximity Competitive Hybridization Assay for the Determination of Phosphorothioate Antisense Oligonucleotide Plasma Concentrations in a Toxicokinetic Study

A novel scintillation proximity competitive hybridization assay was developed for determining plasma concentrations of compound 4003W94, a 15-base phosphorothioate antisense deoxyribonucleotide that is currently under preclinical evaluation for the treatment of restenosis following coronary artery angioplasty. The principle of the assay involves the hybridization binding of antisense (4003W94) to a biotinylated sense oligonucleotide to form a double-stranded nucleic acid complex on the surface of scintillation proximity beads derivatized with streptavidin. As in a competitive radioimmunoassay, there is an inverse relationship between the amount of radioactivity in the final binding complex and the amount of 4003W94 present in the sample being analyzed. Because this is a homogenous assay, no physical separation of bound from free radioligand is necessary. Conventional cross-reactivity studies with either 3′- or 5′-deletion oligomers of 4003W94 indicated that cross-reactivity generally decreased with each base deletion. The assay was used to determine plasma concentrations of 4003W94 equivalents in rhesus monkeys during an exploratory 14-day toxicity study. This method conceivably could be adapted for use as an effectivein vitroscreening tool for the identification of potential antisense oligonucleotide drug candidates or as a diagnostic tool for the detection of pathological disorders.

小腸オリゴペプチド輸送担体クローンを利用したβ‐ラクタム抗生物質の経口デリバリーのスクリーニング系開発への試み

Complementary DNA clone encoding the rat small-intestinal oligopeptide transporter, rat PepT1, was isolated from a rat jejunal cDNA library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicated that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Hydropathy analysis reveals 12 putative transmembrane domains, with a long (204 amino acids) hydrophilic segment containing five potential N-glycosylation sites between transmembrane domains 9 and 10, in the predicted rat protein. Northern blot analysis detected rat PepTl mRNA in the small intestine and kidney. Complementary RNA (cRNA) of PepT1 was synthesized in vitro transcription and injected into Xenopus laevis oocytes to evaluate its transport function. Uptake of a dipeptide, [14C] glycylsarcosine by the oocytes was about 10-times higher than that by oocytes injected with water. The cRNA induced-uptake was enhanced in the presence of an inwardly directed proton gradient and was specifically inhibited by dipeptides and tripeptide, whereas their constitutive amino acids, tetra- and penta-peptide were not inhibitory. Accordingly, the isolated clone was confirmed as a rat intestinal proton-coupled oligopeptide transporter, PepT1. Zwitterionic and dianionic cephalosporins, cefadroxil and ceftibuten, were also specifically taken up by the oocytes injected with cRNA, Furthermore, mutual inhibitory effects on the uptakes were observed between glycylsarcosine and these cephalosporins. Hybrid depletion of the expression of rat or rabbit PepT1 in Xenojus laevis oocytes injected with intestinal total mRNA was studied using antisense oligonucleotides corresponding to the 5'-coding region of PepT1. In oocytes injected with both mRNA hybridized with each antisense oligonucleotide, ceftibuten uptakes were almost completely abolished. However, these uptake were not influenced with respective sense oligonucleotide. These results suggest that PepT1 functions for the intestinal absorption of oligopeptides and, β-lactam antibiotics.

Anti-minK Antisense Decreases the Amplitude of the Rapidly Activating Cardiac Delayed Rectifier K + Current

The rapidly and slowly activating delayed rectifier K+ currents (IKr and IKs, respectively), which have different physiological properties have been identified in cardiac cells from several species, including humans. Although expression of the minimal K+ channel protein (minK) cDNA in some systems results in a current resembling IKs, the role of this gene product in channel function remains controversial. In atrial tumor myocytes (AT-1 cells), no IKs is recorded, but minK mRNA is detected, raising the possibility that expression of the minK gene serves an as-yet-unidentified function. In these experiments, AT-1 cells were exposed to antisense oligonucleotides targeting the 5' translation start site of the minK cDNA cloned from an AT-1 library. Cell size, IKr, and L-type and T-type Ca2+ currents were measured 24 to 48 hours after exposure and compared with data in cells exposed to the corresponding sense oligonucleotide or grown in medium only. Antisense oligonucleotide significantly reduced IKr compared with sense and medium-only control cells in 0 of 2 experiments (n = 3 to 6 cells per treatment in each experiment) at 50 nmol/L, 1 of 2 at 250 nmol/L, 6 of 6 at 1000 nmol/L, and 2 of 2 at 10,000 nmol/L. At 1000 nmol/L, maximum tail current in antisense-exposed cells was 2.5 +/- 0.1 pA/pF (mean +/- SEM, n = 28, 6 separate experiment), 6.6 +/- 0.4 pA/pF in sense-exposed cells (n = 27), 5.4 +/- 0.6 pA/pF in medium-only cells (n = 21), and 5.8 +/- 0.7 pA/pF in cells exposed to a random oligonucleotide (n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclin D3 Is Essential for Megakaryocytopoiesis

A normal cell cycle in most eukaryotic cells consists of a tightly regulated sequence of phases including DNA synthesis (S) followed by a gap (G2), mitosis (M), and a gap (G1). In the megakaryocytic lineage, the cells undergo endomitosis, which involves DNA synthesis in the absence of mitosis, thus giving rise to polyploid cells. We aimed at defining whether the megakaryocytic cell cycle consists of a continuous S phase or of G1/S phases and at determining which cyclins are involved in this process. Studies were performed in primary cultures of mouse bone marrow cells. DNA synthesis in megakaryocytes was followed by determining incorporation of a DNA precursor, bromodeoxyuridine (BrdU), into the cells by in situ staining for BrdU. These experiments showed that no more than 15% of the recognizable megakaryocytes in normal bone marrow are in the process of endomitosis, including S phases interrupted by short gaps. Using immunohistochemistry, we showed that mature megakaryocytes express the G1 phase cyclin and cyclin D3, but not the mitotic cyclin, cyclin B1. Under culture conditions that selectively promote megakaryocytopoiesis, antisense oligonucleotides designed to suppress cyclin D3 expression, but not sense oligonucleotides or antisense oligonucleotides to cyclin B1, dramatically suppress endomitosis and abrogate megakaryocyte development. Our results indicate that endoreduplication in megakaryocytes is associated with low levels of or the absence of cyclin B1, whereas progression through this process depends on the G1 phase for which cyclin D3 is crucial.

106 Chemosensitisation of human adenocarcinoma cells by antisense against the multidrug resistance (MDR1) gene

The MDRI gene encodes P-glycoprotein (Pgp) which pumps cytotoxic agents out of cells and thus induces chemoresistance. Inhibition of this gene could impair Pgp expression and sensitise the cells to cytotoxic drugs. Pgp expression (by Western blot), sensitivity to doxorubicin (by MTT assay) and efflux of the Pgp mediated substrata rhodamine were investigated in human (Hap 2A) adenocarcinoma cells before and after treatment by antisense (AS) and sense (S) oligonucleotides against MDRl. Both ASI (against initiation region) and AS2 (against loop forming site) significantly (approx. 50%) inhibited Pgp expression and gave a 10-fold increase in chemosensitivity to doxorubicin. Both ASI and AS2 inhibited rhodamine efflux. AS2 appeared more effective in all the tests. PgP expression returned to normal within 5 days of withdrawal of AS. Sense oligonucleotides S1 and S2 were ineffective. <h3>Conclusions</h3> MDRI antisense inhibits Pgp expression and sensitises human adenocarcinoma cells (Hep 2A) to doxorubicin chemotherapy.

Volume-sensitive Chloride Channel Activity Does Not Depend on Endogenous P-glycoprotein

To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells.

Reversal of Drug Sensitivity in MDR Subline of P388 Leukemia by Gene‐Targeted Antisense Oligonucleotide

We attempted to reverse multidrug resistance (MDR) by treatment with 25‐mer antisense phosphorothioate oligonucleotide. The phosphorothioate analogs, the sequences of which are sense or antisense to the initiation codon of mousemdr1 mRNA, were tested against murine leukemic P388/S and adriamycin‐resistant P388/ADR cell lines. A weak inhibitory effect on the growth of P388/S and P388/ADR cells was observed at a sense and antisense oligonucleotide concentration of 30 μM. Using the monoclonal antibody to P‐glycoprotein and a flow cytometry technique, we showed that the level of expression of P‐glycoprotein in P388/ADR cells treated with antisense oligonucleotide was lower than when treated with sense oligonucleotide. The antisense oligonucleotide potentiated the growth‐inhibitory effect of vinblastine on P388/ADR cells, whereas sense oligonucleotide did not. This was accompanied by an increase in vinblastine retention in the cells. The reversal of the resistance by antisense oligonucleotide was increased by the combination with 1 μM verapamil. These results suggest that the antisense oligonucleotide and low dose verapamil may be useful in circumventing the resistance to anticancer drugs of MDR tumors.