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Biomedical subjects

N M Dean

Publications and source records attributed to N M Dean.

At least 37 records · Page 2Linked to original sources
PubMed2000

Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis.

Aberrant apoptosis-mediated cell death is believed to result in a number of different human diseases. For example, excessive apoptosis in the liver can result in fulminant and autoimmune forms of hepatitis. We have explored the possibility that inhibition of Fas expression in mice would reduce the severity of fulminant hepatitis. To do this, we have developed a chemically modified 2'-O-(2-methoxy)ethyl antisense oligonucleotide (ISIS 22023) inhibitor of mouse Fas expression. In tissue culture, this oligonucleotide induced a reduction in Fas mRNA expression that was both concentration- and sequence-specific. In Balb/c mice, dosing with ISIS 22023 reduced Fas mRNA and protein expressions in liver by 90%. The ID50 for this response was 8-10 mg kg-1 daily dosing, and the reduction was highly dependent on oligonucleotide sequence, oligonucleotide concentration in liver, and treatment time. Pretreatment with ISIS 22023 completely protected mice from fulminant hepatitis induced by agonistic Fas antibody, by a mechanism entirely consistent with an oligonucleotide antisense mechanism of action. In addition, oligonucleotide-mediated suppression of Fas expression reduced the severity of acetaminophen-mediated fulminant hepatitis, but was without effect on concanavalin A-mediated hepatitis. Our results demonstrate that 2'-O-(2-methoxy)ethyl containing antisense oligonucleotides targeting Fas can exert in vivo pharmacological activity in liver, and suggest that oligonucleotide inhibitors of Fas may be useful in the treatment of human liver disease.

Animals↗
PubMed2000

Antisense inhibition of membrane-bound human interleukin-5 receptor-alpha chain does not affect soluble receptor expression and induces apoptosis in TF-1 cells.

Binding of human interleukin-5 (HuIL-5) to its membrane-anchored receptor (IL-5R) triggers multiple signaling pathways, cellular proliferation, and maturational responses, as well as protection from apoptosis. In contrast, soluble forms of the HuIL-5R have been shown to inhibit IL-5 signaling and, therefore, may represent naturally occurring negative regulators of IL-5 function. Because of the central role of IL-5 in promoting eosinophilia and airway hyperresponsiveness in animal models of asthma, antisense oligonucleotides specific either for the membrane form alone or for sequences shared between both the membrane and soluble forms of the HuIL-5Ralpha ligand binding chain were designed. The activities of these oligonucleotides were characterized in IL-5R-expressing erythroleukemic TF-1 cells. Herein we report that an antisense oligonucleotide targeted to a sequence unique to the alternatively spliced membrane-bound form of the HuIL-5Ralpha chain has been developed that selectively inhibits membrane, but not soluble, mRNA isoform expression. Both this membrane-specific oligonucleotide and an antisense oligonucleotide targeted to sequence common to both membrane and soluble isoforms were found to potently suppress cell surface IL-5Ralpha levels and IL-5-mediated cell survival by inducing apoptosis similar to IL-5 withdrawal. Thus, these oligonucleotides represent unique genetic agents with therapeutic potential for diseases with an eosinophilic component.

Alternative Splicing↗
PubMed2000

Deletion of individual exons and induction of soluble murine interleukin-5 receptor-alpha chain expression through antisense oligonucleotide-mediated redirection of pre-mRNA splicing.

Expression of the interleukin-5 receptor-alpha (IL-5Ralpha) chain is thought to play an important role in the pathogenesis of asthma and other eosinophilic diseases. With antisense oligonucleotides (ASOs) chemically modified to provide increased hybridization affinity for RNA but that do not support RNase H-mediated cleavage (2'-O-methoxyethyl-modified ASOs), we show that constitutive splicing of murine IL-5Ralpha mRNA can be modulated in cells such that individual exons may be selectively deleted from mature transcripts. Specific deletion of individual exons and redirection of alternative splicing of the IL-5Ralpha mRNA have been achieved with this approach, by targeting 3'-splice sites or exon sequences immediately downstream of an alternative splice site. ASO targeting with these strategies resulted in inhibition of mRNA and protein levels of the membrane IL-5Ralpha isoform capable of signaling IL-5-mediated growth and antiapoptotic signals to eosinophils. Membrane isoform IL-5Ralpha inhibition was coupled with an increase in expression of mRNA for the alternatively spliced soluble isoform, which binds IL-5 extracellularly and may block its function. These observations suggest the potential general therapeutic use of an antisense approach to increase expression of variant RNA transcripts and to thereby produce proteins devoid of specific functional domains that may impact disease processes, as well as its specific utility for modulating expression of a key cytokine receptor implicated in allergic inflammation.

Alternative Splicing↗
PubMed2000

5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

3T3 Cells↗
PubMed2000

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner.

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.

Apoptosis↗
PubMed1999

Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli.

The epidermis is continually exposed to harmful mutagens that have the potential to cause DNA damage. To protect the skin from accumulating mutated cells, keratinocytes have developed a highly regulated mechanism of eliminating damaged cells through apoptosis. Bcl-xL is a well-described cell survival protein that when overexpressed in skin can protect keratinocytes from UV radiation-induced apoptosis. To begin to unravel the complex mechanisms that keratinocytes use to survive, we wanted to characterize the role of endogenous Bcl-xL in protecting cells from death. In this study, we describe the development and characterization of an antisense inhibitor to Bcl-xL. We show that this inhibitor reduces Bcl-xL RNA and protein in a concentration-dependent, sequence-specific manner. Furthermore, treatment of keratinocytes and epithelial cells with this inhibitor sensitizes these cells to UV-B radiation and cisplatinum treatment-induced apoptosis. Thus, these results offer direct evidence that Bcl-xL is critical in the protection of skin and epithelial cells from apoptosis and provide a basis for the role of Bcl-xL in keratinocyte and epithelial cell survival.

Apoptosis↗
PubMed1999

Ser/Thr protein phosphatase type 5 (PP5) is a negative regulator of glucocorticoid receptor-mediated growth arrest.

Ligand-induced glucocorticoid receptor (GR) activation has recently been linked to the inhibition of cell proliferation via the transcriptional induction of p21(WAF1/Cip1), which functions as a universal inhibitor of cyclin-dependent protein kinases. Herein, we identify a Ser/Thr protein phosphatase (PP5) that promotes cellular proliferation by inhibiting both glucocorticoid and p53-mediated signaling pathways leading to p21(WAF1/Cip1)-mediated growth arrest. The suppression of PP5 expression (1) markedly increases the association of GR with its cognate DNA-binding sequence, (2) induces GR transcriptional activity without the addition of hormone, and (3) increases dexamethasone-mediated induction of GR reporter activity to a level that is approximately 10 times greater than the maximal response obtainable in the presence of PP5. PP5 has no apparent effect on the binding of hormone to the GR, and dexamethasone-mediated growth arrest correlates with an increase in p53 phosphorylation. Comparative studies in p53-wild-type, p53-defective, and p53-deficient cell lines indicate that either (1) p53 participates in GR-mediated induction of p21(WAF1/Cip1), with the hyperphosphorylation of basal p53 induced by glucocorticoids sufficient for the propagation of an antiproliferative response when PP5 expression is inhibited, or (2) PP5 acts where p53-mediated and GR-induced signaling networks converge to regulate the transcriptional induction of p21(WAF1/Cip1). Thus, aberrant PP5 expression may have an additive effect on the development of human cancers by promoting cell proliferation via the inhibition of a GR-induced antiproliferative signaling cascade, and facilitating neoplastic transformation via the inhibition of a growth-arresting p53-mediated response that guards against genomic instability.

Binding Sites↗
PubMed1999

Up-regulation of the Pit-2 phosphate transporter/retrovirus receptor by protein kinase C epsilon.

The membrane receptors for the gibbon ape leukemia retrovirus and the amphotropic murine retrovirus serve normal cellular functions as sodium-dependent phosphate transporters (Pit-1 and Pit-2, respectively). Our earlier studies established that activation of protein kinase C (PKC) by treatment of cells with phorbol 12-myristate 13-acetate (PMA) enhanced sodium-dependent phosphate (Na/Pi) uptake. Studies now have been carried out to determine which type of Na/Pi transporter (Pit-1 or Pit-2) is regulated by PKC and which PKC isotypes are involved in the up-regulation of Na/Pi uptake by the Na/Pi transporter/viral receptor. It was found that the activation of short term (2-min) Na/Pi uptake by PMA is abolished when cells are infected with amphotropic murine retrovirus (binds Pit-2 receptor) but not with gibbon ape leukemia retrovirus (binds Pit-1 receptor), indicating that Pit-2 is the form of Na/Pi transporter/viral receptor regulated by PKC. The PKC-mediated activation of Pit-2 was blocked by pretreating cells with the pan-PKC inhibitor bisindolylmaleimide but not with the conventional PKC isotype inhibitor Gö 6976, suggesting that a novel PKC isotype is required to regulate Pit-2. Overexpression of PKCepsilon, but not of PKCalpha, -delta, or -zeta, was found to mimic the activation of Na/Pi uptake. To further establish that PKCepsilon is involved in the regulation of Pit-2, cells were treated with PKCepsilon-selective antisense oligonucleotides. Treatment with PKCepsilon antisense oligonucleotides decreased the PMA-induced activation of Na/Pi uptake. These results indicate that PMA-induced stimulation of Na/Pi uptake by Pit-2 is specifically mediated through activation of PKCepsilon.

3T3 Cells↗
PubMed1999

Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression.

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.

Base Sequence↗
PubMed1999

Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides.

Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of antiapoptotic to proapoptotic proteins. By manipulating levels of these proteins, cells can become sensitized to undergo apoptosis in response to chemotherapeutic agents. Alternative splicing of the bcl-x gene gives rise to two proteins with antagonistic functions: Bcl-xL, a well-characterized antiapoptotic protein, and Bcl-xS, a proapoptotic protein. We show here that altering the ratio of Bcl-xL to Bcl-xS in the cell using an antisense oligonucleotide permitted cells to be sensitized to undergo apoptosis in response to ultraviolet B radiation and chemotherapeutic drug treatment. These results demonstrate the ability of a chemically modified oligonucleotide to alter splice site selection in an endogenous gene and illustrate a powerful tool to regulate cell survival.

Alternative Splicing↗
PubMed1999

Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells.

1. Thymidylate synthase (TS), the key enzyme in de novo synthesis of thymidine, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide down-regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), and the folate analogue Tomudex (ICI D1694; N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theon yl-L-glutamic acid). 2. 2'-Methoxyethoxylated, phosphorothioated 20-mer oligodeoxynucleotides (ODNs), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomized order, against which all the biological effects were compared. Following a 6-h transfection of HeLa cells using polycationic liposome at 3 microg ml(-1), ODN 83 (50 nM), complementary to a region in the 3'-untranslated region of the TS mRNA, decreased TS mRNA levels by approximately 70% within 24 h. ODN 83 also decreased TS enzyme activity, as measured by binding of TS to radiolabelled 5-fluorodeoxyuridine monophosphate. In addition to inhibiting proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxicity of Tomudex or 5-FU, added 1 day following transfection, by 50 - 60%. ODN 83 also enhanced sensitivity to 5-FUdR by 70%, but did not affect the toxicity of cisplatin, chlorambucil, melphalan, doxorubicin, ionizing radiation, paclitaxel, or irinotecan. 3. These data indicate that antisense ODN down-regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS-targeted drugs.

Antimetabolites, Antineoplastic↗
PubMed1999

Induction of p53-dependent, insulin-like growth factor-binding protein-3-mediated apoptosis in glioblastoma multiforme cells by a protein kinase Calpha antisense oligonucleotide.

Protein kinase Calpha (PKCalpha) expression is related to tumor progression in glioblastoma multiforme (GBM), the most common malignant brain tumor in adults. To determine whether PKCalpha regulates an anti-apoptotic survival pathway in GBM, A172 GBM cells were treated with a PKCalpha-selective antisense oligonucleotide. PKCalpha antisense oligonucleotide treatment was accompanied by reduction in PKCalpha levels and the induction of wild-type p53 and insulin-like growth factor-binding protein-3 (IGFBP3) 24-72 h after treatment, a period that coincided with the appearance of apoptotic cell death as detected by DNA fragmentation. There were no significant changes in the levels of Bcl-XL, Bax, and p21(WAF1). Induction of p53 after PKCalpha down-regulation was not associated with increased mRNA expression, but increased IGFBP3 levels were accompanied by increased mRNA levels. Recombinant human IGFBP3 induced an apoptotic effect that was similar to the PKCalpha antisense oligonucleotide, and its effect was blocked by IGF-I. These results suggest that one mechanism by which PKCalpha produces its antiapoptotic activity in GBM cells is by suppressing the p53-mediated activation of IGFBP3.

Apoptosis↗
PubMed1999

The Jun kinase 2 isoform is preferentially required for epidermal growth factor-induced transformation of human A549 lung carcinoma cells.

We have previously found that epidermal growth factor (EGF) mediates growth through the Jun N-terminal kinase/stress-activated kinase (JNK/SAPK) pathway in A549 human lung carcinoma cells. As observed here, EGF treatment also greatly enhances the tumorigenicity of A549 cells, suggesting an important role for JNK in cancer cell growth (F. Bost, R. McKay, N. Dean, and D. Mercola, J. Biol. Chem. 272:33422-33429, 1997). Several isoforms families of JNK, JNK1, JNK2, and JNK3, have been isolated; they arise from alternative splicing of three different genes and have distinct substrate binding properties. Here we have used specific phosphorothioate oligonucleotides targeted against the two major isoforms, JNK1 and JNK2, to discriminate their roles in EGF-induced transformation. Multiple antisense sequences have been screened, and two high-affinity and specific candidates have been identified. Antisense JNK1 eliminated steady-state mRNA and JNK1 protein expression with a 50% effective concentration (EC50) of <0.1 microM but did not alter JNK2 mRNA or protein levels. Conversely, antisense JNK2 specifically eliminated JNK2 steady-state mRNA and protein expression with an EC50 of 0.1 microM. Antisense JNK1 and antisense JNK2 inhibited by 40 and 70%, respectively, EGF-induced total JNK activity, whereas sense and scrambled-sequence control oligonucleotides had no effect. The elimination of mRNA, protein, and JNK activities lasted 48 and 72 h following a single Lipofectin treatment with antisense JNK1 and JNK2, respectively, indicating sufficient duration for examining the impact of specific elimination on the phenotype. Direct proliferation assays demonstrated that antisense JNK2 inhibited EGF-induced doubling of growth as well as the combination of active antisense oligonucleotides did. EGF treatment also induced colony formation in soft agar. This effect was completely inhibited by antisense JNK2 and combined-antisense treatment but not altered by antisense JNK1 alone. These results show that EGF doubles the proliferation (growth in soft agar as well as tumorigenicity in athymic mice) of A549 lung carcinoma cells and that the JNK2 isoform but not JNK1 is utilized for mediating the effects of EGF. This study represents the first demonstration of a cellular phenotype regulated by a JNK isoform family, JNK2.

Agar↗
PubMed1999

Preclinical and clinical pharmacology of antisense oligonucleotides.

Antisense oligonucleotides are short (typically 15-20 bases in length pieces of synthetically manufactured, chemically modified DNA or RNA. They are designed to interact by Watson-Crick base pairing with mRNA transcripts encoding proteins of interest, and by virtue of this interaction inhibit the expression of the protein encoded in the mRNA. Since their first proposed use in 1978, antisense oligonucleotides have become come widely used as tools to modulate gene expression in tissue culture. The great specificity that these compounds exhibited in vitro has also led them to be viewed as potentially therapeutically useful. This interest has resulted in the progression of (to date) a dozen compounds into human clinical trials for a variety of indications ranging from cancer to inflammatory diseases. Here, we will review some of the progress that has been made with antisense pharmacology, both in vitro and in vivo, as well as describe the current status of this class of compound in clinical trials.

Clinical Trials as Topic↗
PubMed1998

Human melanoma metastasis is inhibited following ex vivo treatment with an antisense oligonucleotide to protein kinase C-alpha.

To determine whether alteration of PKC alpha expression would affect the metastatic potential of human melanoma cells, replicate cultures of C8161 cells were treated in vitro with a phosphorothioate antisense oligodeoxynucleotide (ODN) that specifically inhibits PKC alpha expression (ISIS-3521). Control C8161 cultures were treated with a scrambled sequence ODN, cationic liposomes or were left untreated. Northern blots demonstrated 70% inhibition of PKC alpha mRNA in ISIS-3521-treated cells compared to controls. Metastasis was suppressed by 75% when ISIS-3521-treated cells were injected intravenously into athymic mice. These results show that PKC alpha expression is important in the regulation of human melanoma metastasis.

Animals↗
PubMed1998

Serine/threonine protein phosphatase type 5 acts upstream of p53 to regulate the induction of p21(WAF1/Cip1) and mediate growth arrest.

Understanding how alterations in growth control pathways are translated into changes in the cell cycle regulatory machinery is a major challenge for understanding the development of human cancers. The ability of both tumor suppressor proteins, p53 and BRCA1, to induce the expression of p21(WAF1/Cip1) in combination with the inhibitory activity of p21(WAF1/Cip1) against cyclin-dependent kinases suggests that the regulation of p21(WAF1/Cip1) expression is an important aspect of mammalian cell cycle growth control. To elucidate the role of serine/threonine protein phosphatase type 5 (PP5) in processes regulating cell cycle progression, we developed antisense oligodeoxynucleotides targeted against PP5 (e.g. ISIS 15534) that specifically inhibit PP5 gene expression. Employing ISIS 15534, we demonstrate that the specific inhibition of PP5 gene expression has a marked antiproliferative effect on cells, characterized by induction of p21(WAF1/Cip1) and the subsequent arrest of cell growth. Investigations into the mechanisms leading to growth arrest reveal that, in the absence of PP5, the expression of p21(WAF1/Cip1) is induced in p53-competent A549 cells but not in p53 protein-deficient T-24 cells. Employing a stable cell line derived from p53-deficient human fibroblast that contains tetracycline-regulated transactivator and operator plasmids to control the expression of wild-type p53 (TR9-7 cells), we then show that the induction of p21(WAF1/Cip1), which occurs in response to the inhibition of PP5 expression, requires the p53 protein. Additional studies indicate that PP5 acts upstream of p53, influencing both the phosphorylation state and the ability of p53 to bind DNA, without causing an increase in p53 gene transcription. Together these studies suggest that PP5 is a regulatory component of a signaling pathway that affords replicating cells G1 checkpoint growth control and that it is the regulation of PP5 that, in turn, controls p53-mediated expression of p21(WAF1/Cip1) and growth arrest in this pathway. In addition, since the inhibition of PP5 gene expression has marked antiproliferative activity and the overexpression of p21(WAF1/Cip1) blocks the growth of tumor cells, these studies suggest that compounds that inhibit of PP5 gene expression may be useful in the treatment of human cancers.

Cell Division↗
PubMed1998

Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus.

Antisense oligonucleotides complementary to specific mRNA sequences are widely used inhibitors of gene expression in vitro and in vivo . In vitro cationic lipids have been demonstrated to increase the pharmacological activity of antisense oligonucleotides by increasing cellular uptake and facilitating nuclear accumulation. We have investigated the intracellular fate of oligonucleotide/cationic lipid complexes using fluorescently labeled lipids and oligonucleotides targeted to protein kinase C-alpha. After addition to cells the lipids initially co-localized with the oligonucleotide on the cell surface and in fine punctate structures within the cytoplasm. At later times the oligonucleotide began to accumulate in the nucleus as well as the cytoplasm. In contrast, the cationic lipid remained localized to the cell surface and the cytoplasm and was never found in the nucleus. Expression of protein kinase C-alpha mRNA did not begin to decline until after oligonucleotide was seen in the nucleus. This was also coincident with the transient appearance of a smaller mRNA transcript believed to result from RNase H cleavage of protein kinase C-alpha mRNA. These data suggest that although cationic lipids facilitate uptake of oligonucleotides, the complex must disassociate before the oligonucleotide can gain access to the nucleus and induce degradation of targeted mRNA.

Boron Compounds↗