Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Overexpression of a Kinase-deficient Transforming Growth Factor-β Type II Receptor in Mouse Mammary Stroma Results in Increased Epithelial Branching

Members of the transforming growth factor-β (TGF-β) superfamily signal through heteromeric type I and type II serine/threonine kinase receptors. Transgenic mice that overexpress a dominant-negative mutation of the TGF-β type II receptor (DNIIR) under the control of a metallothionein-derived promoter (MT-DNIIR) were used to determine the role of endogenous TGF-βs in the developing mammary gland. The expression of the dominant-negative receptor was induced with zinc and was primarily localized to the stroma underlying the ductal epithelium in the mammary glands of virgin transgenic mice from two separate mouse lines. In MT-DNIIR virgin females treated with zinc, there was an increase in lateral branching of the ductal epithelium. We tested the hypothesis that expression of the dominant-negative receptor may alter expression of genes that are expressed in the stroma and regulated by TGF-βs, potentially resulting in the increased lateral branching seen in the MT-DNIIR mammary glands. The expression of hepatocyte growth factor mRNA was increased in mammary glands from transgenic animals relative to the wild-type controls, suggesting that this factor may play a role in TGF-β-mediated regulation of lateral branching. Loss of responsiveness to TGF-βs in the mammary stroma resulted in increased branching in mammary epithelium, suggesting that TGF-βs play an important role in the stromal–epithelial interactions required for branching morphogenesis.

Distinct Endocytic Responses of Heteromeric and Homomeric Transforming Growth Factor β Receptors

Transforming growth factor β (TGFβ) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGFβ receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGFβ receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor α or β receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGFβ receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGFβ receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGFβ receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGFβ receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGFβ receptors and that TGFβ receptor heteromers and homomers show distinct trafficking behavior.

Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

We are studying endoplasmic reticulum–associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ∼45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332 in which the single used N-glycosylation consensus site had been removed (RI332-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI332. After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332 and RI332-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI332 and RI332-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

Anti-β2 glycoprotein I (β2GPI) autoantibodies recognize an epitope on the first domain of β2GPI

Anticardiolipin (aCL) autoantibodies are associated with thrombosis, recurrent fetal loss, and thrombocytopenia. Only aCL found in autoimmune disease require the participation of the phospholipid binding plasma protein β2 glycoprotein I (β2GPI) for antibody binding and now are called anti-β2GPI. The antigenic specificity of aCL affinity purified from 11 patients with high titers was evaluated in an effort to better understand the pathophysiology associated with aCL. Seven different recombinant domain-deleted mutants of human β2GPI, and full length human β2GPI (wild-type), were used in competition assays to inhibit the autoantibodies from binding to immobilized wild-type β2GPI. Only those domain-deleted mutants that contained domain 1 inhibited the binding to immobilized wild-type β2GPI from all of the patients. The domain-deleted mutants that contained domain 1 inhibited all aCL in a similar but not identical pattern, suggesting that these aCL recognize a similar, but distinguishable, epitope(s) present on domain 1.

FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor β (TGF-β) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12−/−) mice manifest cell cycle arrest in G1 phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-β1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-β receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-β receptor construct. TGF-β receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12−/− cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12−/− cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-β receptor signaling.

Brain 5α-dihydroprogesterone and allopregnanolone synthesis in a mouse model of protracted social isolation

Allopregnanolone (ALLO), is a brain endogenous neurosteroid that binds with high affinity to γ-aminobutyric acid type A (GABAA) receptors and positively modulates the action of GABA at these receptors. Unlike ALLO, 5α-dihydroprogesterone (5α-DHP) binds with high affinity to intracellular progesterone receptors that regulate DNA transcription. To investigate the physiological roles of ALLO and 5α-DHP synthesized in brain, we have adopted a mouse model involving protracted social isolation. In the frontal cortex of mice, socially isolated for 6 weeks, both neurosteroids were decreased by approximately 50%. After administration of (17β)-17-(bis-1-methyl amino carbonyl) androstane-3,5-diene-3-carboxylic acid (SKF105,111), an inhibitor of the enzyme (5α-reductase Type I and II) that converts progesterone into 5α-DHP, the ALLO and 5α-DHP content of frontal cortex of both group-housed and socially isolated mice decreased exponentially to 10%–20% of control values in about 30 min. The fractional rate constants (k h−1) of ALLO and 5α-DHP decline multiplied by the ALLO and 5α-DHP concentrations at any given steady-state estimate the rate of synthesis required to maintain that steady state. After 6 weeks of social isolation, ALLO and 5α-DHP biosynthesis rates were decreased to 30% of the values calculated in group-housed mice. Moreover, in socially isolated mice, the expression of 5α-reductase Type I mRNA and protein was approximately 50% lower than in group-housed mice whereas 3α-hydroxysteroid oxidoreductase mRNA expression was equal in the two groups. Protracted social isolation in mice may provide a model to investigate whether 5α-DHP by a genomic action, and ALLO by a nongenomic mechanism down-regulate the action of drugs acting as agonists, partial agonists, or positive allosteric modulators of the benzodiazepine recognition sites expressed by GABAA receptors.

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

In skeletal muscle, transcription of the gene encoding the mouse type Iα (RIα) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RIα protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIα or RIIα fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIα subunits and requires the amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the full-length RIα–green fluorescent protein template abolishes localization, indicating that dimerization of RIα is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIα at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Iα homologue RCE with AKAPCE and for in vitro binding of RIα to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RIα tethering at this site.

A DAF-1-binding protein BRA-1 is a negative regulator of DAF-7 TGF-β signaling

We have identified homologs of a human BMP receptor-associated molecule BRAM1 in Caenorhabditis elegans. One of them, BRA-1, has been found to bind DAF-1, the type I receptor in the DAF-7 transforming growth factor-β pathway through the conserved C-terminal region. As analyzed using a BRA-1∷GFP (green fluorescent protein) fusion gene product, the bra-1 gene is expressed in amphid neurons such as ASK, ASI, and ASG, where daf-1 is also expressed. A loss-of-function mutation in bra-1 exhibits robust suppression of the Daf-c phenotype caused by the DAF-7 pathway mutations. We propose that BRA-1 represents a novel class of receptor-associated molecules that negatively regulate transforming growth factor-β pathways.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of ≈40 nitr genes are contiguous in the genome and span ≈0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.

Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.

In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of human immunodeficiency virus type I Rev, were selected from a random sequence RNA pool. Several of the selected RNAs could bind the free peptide more tightly than a natural RNA ligand, the Rev-binding element. In accord with the hypothesis that protein and nucleic acid binding cusps are functionally similar, interactions between aptamers and the peptide target could be disrupted by sequence substitutions. Moreover, the aptamers appeared to be able to bind peptides with different solution conformations, implying an induced fit mechanism for binding. Just as anti-peptide antibodies can sometimes recognize the corresponding epitope when presented in a protein, the anti-peptide aptamers were found to specifically bind to Rev.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.