Sequence-based discovery of a synthetic peptide inhibitor of caspase 6.

We recently demonstrated that tumor necrosis factor alpha activates caspase 6, which in turn cleaves transcription factor AP-2 alpha. We mapped the cleavage site at 19 amino acids from the N-terminus at the sequence aspartate-argenine-histidine-aspartate (DRHD). Mutating aspartic acid at position 19 abrogated the cleavage site. From these observations, we hypothesized that the DRHD peptide could act as a caspase 6 inhibitor. To test this hypothesis, the peptide zAsp(Ome)-Arg-His-Asp(Ome)-fluoromethyl ketone (zDRHDfmk) was synthesized. Here we show that zDRHDfmk inhibits TNFalpha-induced caspase 6 activity and apoptosis in breast cancer cells. When compared to other caspase inhibitors, zDRHDfmk inhibited caspase 6 activity more effectively than the general caspase inhibitor zVal-Ala-Lys(Ome)-fluoromethy ketone (zVADfmk) or the caspase 6 inhibitor zVal-Glu-Ile-Asp-(Ome)-fluoromethyl ketone (zVEIDfmk). However, it was less effective in inhibiting TNFalpha-induced apoptosis than zVADfmk or zVEIDfmk, presumably because caspase 6 is only one of at least three effector caspases, the others being caspase 3 and 7, that are active during caspase-dependent apoptosis. The discovery of this sequence-based caspase 6 inhibitor provides a new tool for studying caspase 6. More importantly, it could be used, in combination with other agents, as a drug to inhibit apoptosis in neurodegenrative diseases such as Alzheimer's, Parkinson and amyotrophic lateral sclerosis.

An MMP-2/MMP-9 inhibitor, 5a, enhances apoptosis induced by ligands of the TNF receptor superfamily in cancer cells.

Several studies have shown that matrix metalloproteases (MMPs) promote tumor growth, invasion, and metastasis. Consequently, MMP inhibitors have been developed as a new class of anticancer drugs, many of which are in clinical trials. The exact mechanism of the antineoplastic activity of MMP antagonists is unknown. To investigate the mechanism, we hypothesized that MMP inhibitors enhance the actions of apoptosis-inducing agents. To test this hypothesis, we treated breast, melanoma, leukemia, osteosarcoma, and normal breast epithelial cells with (2R)-2-[(4-biphenylsulfonyl)amino]-3-phenylproprionic acid (compound 5a), an organic inhibitor of MMP-2/MMP-9, alone or in combination with TNFalpha or other apoptotic agents. FACS analysis showed that 5a interacted synergistically with ligands of the TNF receptor superfamily, including TNFalpha and TNF receptor-like apoptosis-inducing ligand (TRAIL), and with a Fas-cross-linking antibody (CH11), UV, paclitaxel, thapsigargin, and staurosporin, to induce apoptosis in a cell-type-specific manner. Other MMP inhibitors did not synergize with TNFalpha. Compound 5a did not act directly on the mitochondrion or via changes in protein synthesis. Instead, the mechanism requires ligand-receptor interaction and caspase 8 activation. Investigation of the effect of 5a on tumor growth in vivo revealed that continuous treatment of subcutaneous melanoma with a combination of 5a plus TRAIL reduced tumor growth and angiogenesis in nude mice. Our data demonstrate that 5a possesses a novel proapoptotic function, thus providing an alternative mechanism for its antineoplastic action. These observations have important implications for combination cancer therapy.

Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11. (AU)

Inhibition of focal adhesion kinase by antisense oligonucleotides enhances the sensitivity of breast cancer cells to camptothecins

This study shows a strong association between cell attachment to substratum and activation of beta 1-integrin-signaling with resistance to the camptothecin derivative topotecan (TPT) in breast cancer cells. We propose a mechanistic-driven approach to sensitize the cells to camptothecins. ZR-75-1 anchorage-dependent breast cancer cell line, its derivative 9D3S suspension cells (9D3S-S), and 9D3S cells attached to fibronectin-coated plates (9D3S-A) were treated with TPT (1 microM) or CPT-11 (40 microM) for 48 h. Programmed cell death (PCD), as shown by poly(ADP-ribose) polymerase (PARP), pro-caspase-3 and pro-caspase-9 cleavage, was observed in 9D3S-S cells but not in ZR-75-1 or 9D3S-A cells. Because p125 focal adhesion kinase (FAK) is a transducer in the beta 1-integrin signaling pathway, it is essential to cell adhesion and it is overexpressed in metastatic breast cancer, we hypothesized that attenuation of FAK might enhance the sensitivity of breast cancer cells to camptothecins. Moreover, inhibition of FAK gene expression by a phosphorothioated antisense oligodeoxynucleotide targeting the portion of the gene encoding amino acids 262-268, increased the sensitivity of ZR-75-1, MDA-MB-231 and MCF7 breast cancer cells to treatment with TPT or CPT-11.

Transcription factor AP-2alpha is preferentially cleaved by caspase 6 and degraded by proteasome during tumor necrosis factor alpha-induced apoptosis in breast cancer cells.

Several reports have linked activating protein 2alpha (AP-2alpha) to apoptosis, leading us to hypothesize that AP-2alpha is a substrate for caspases. We tested this hypothesis by examining the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of AP-2 in breast cancer cells. Here, we provide evidence that TNF-alpha downregulates AP-2alpha and AP-2gamma expression posttranscriptionally during TNF-alpha-induced apoptosis. Both a general caspase antagonist (zVADfmk) and a caspase 6-preferred antagonist (zVEIDfmk) inhibited TNF-alpha-induced apoptosis and AP-2alpha downregulation. In vivo tests showed that AP-2alpha was cleaved by caspases ahead of the DNA fragmentation phase of apoptosis. Recombinant caspase 6 cleaved AP-2alpha preferentially, although caspases 1 and 3 also cleaved it, albeit at 50-fold or higher concentrations. Activated caspase 6 was detected in TNF-alpha-treated cells, thus confirming its involvement in AP-2alpha cleavage. All three caspases cleaved AP-2alpha at asp(19) of the sequence asp-arg-his-asp (DRHD(19)). Mutating D(19) to A(19) abrogated AP-2alpha cleavage by all three caspases. TNF-alpha-induced cleavage of AP-2alpha in vivo led to AP-2alpha degradation and loss of DNA-binding activity, both of which were prevented by pretreatment with zVEIDfmk. AP-2alpha degradation but not cleavage was inhibited in vivo by PS-431 (a proteasome antagonist), suggesting that AP-2alpha is degraded subsequent to cleavage by caspase 6 or caspase 6-like enzymes. Cells transfected with green fluorescent protein-tagged mutant AP-2alpha are resistant to TNF-alpha-induced apoptosis, further demonstrating the link between caspase-mediated cleavage of AP-2alpha and apoptosis. This is the first report to demonstrate that degradation of AP-2alpha is a critical event in TNF-alpha-induced apoptosis. Since the DRHD sequence in vertebrate AP-2 is widely conserved, its cleavage by caspases may represent an important mechanism for regulating cell survival, proliferation, differentiation, and apoptosis.

Transactivation of the urokinase-type plasminogen activator receptor gene through a novel promoter motif bound with an activator protein-2alpha-related factor.

The urokinase receptor overexpressed in invasive cancers promotes laminin degradation. The current study was undertaken to identify cis elements and trans-acting factors activating urokinase receptor expression through a footprinted (-148/-124) region of the promoter containing putative activator protein-2- and Sp1-binding motifs. Mobility shifting experiments using nuclear extract from a high urokinase receptor-expressing cell line (RKO) indicated that Sp1, Sp3, and a factor similar to, but distinct from, activator protein-2alpha bound to this region. Mutations preventing the binding of the activator protein 2alpha-related factor diminished urokinase receptor promoter activity. In RKO cells, the expression of a negative regulator of activator protein-2 function diminished urokinase receptor promoter activity, protein, and laminin degradation. Conversely, urokinase receptor promoter activity in low urokinase receptor-expressing GEO cells was increased by activator protein-2alphaA expression. Although using GEO nuclear extract, little activator protein-2alpha-related factor bound to the footprinted region, phorbol 12-myristate 13-acetate treatment, which induces urokinase receptor expression, increased complex formation. Mutations preventing the activator protein-2alpha-related factor and Sp1/Sp3 binding reduced urokinase receptor promoter stimulation by this agent. Thus, the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the urokinase receptor is mediated partly through trans-activation of the promoter via a sequence (-152/-135) bound with an activator protein-2alpha-related factor.

Proteolytic activity in amyotrophic lateral sclerosis IgG preparations.

Sporadic amyotrophic lateral sclerosis is a motor neuron disease of unknown origin. Autoimmunity against voltage-gated calcium channels is one mechanism hypothesized to be the cause of the disease. In support of this hypothesis, it was previously reported that amyotrophic lateral sclerosis IgG specifically blocked the binding of 8B7 monoclonal antibody to the alpha1 subunit of voltage-gated calcium channels, suggesting overlapping epitopes of the two antibodies. It is, however, possible that the 8B7 epitope was destroyed by proteases. Data presented here show that the blocking of 8B7 binding to the alpha1 subunit by diethylaminoethyl cellulose (DEAE)-purified amyotrophic lateral sclerosis IgG was not observed with Fab fragments of amyotrophic lateral sclerosis IgG. The blocking was prevented by serine protease inhibitors. Moreover, it was reproduced by plasminogen and urokinase. These observations suggest that raised proteolytic activity in amyotrophic lateral sclerosis IgG preparations may be responsible for the blockade of 8B7 monoclonal antibody demonstrated previously. They also indicate the need to be particularly cautious when interpreting the results of incubation in amyotrophic lateral sclerosis sera or IgG preparations. Furthermore, they suggest that proteases may be partly responsible for some of the effects previously described for amyotrophic lateral sclerosis IgG. However, the proteolytic activity needs to be better defined and its possible role in amyotrophic lateral sclerosis investigated.

beta-Amyloid1-40 increases expression of beta-amyloid precursor protein in neuronal hybrid cells.

Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for beta-amyloid peptide (beta-AP), a 40-43-amino-acid peptide derived from a larger membrane glycoprotein, beta-amyloid precursor protein (beta-APP). Previous experiments have demonstrated that beta-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that beta-APP mRNA content is increased 3.5-fold in 24 h after treatment with beta-AP1-40. Accompanying beta-AP1-40-induced cell injury, levels of cell-associated beta-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of beta-APP and 12- and 4-kDa fragments are also increased. Application of beta-APP antisense oligodeoxynucleotide reduces both cytotoxicity and beta-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase beta-APP expression. These results suggest a selective positive feedback mechanism whereby beta-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of beta-APP with the net consequence of further neuronal damage.

Alpha-1 subunits of voltage gated Ca2+ channels in the mesencephalon x neuroblastoma hybrid cell line MES23.5.

The identity of alpha 1 subunits from voltage operated Ca2+ channels was determined in the rat/mouse mesencephalon x N18TG2 hybridoma cell line MES23.5, by sequence analysis of reverse transcription-polymerase chain reaction products and antagonist binding. Sequences were derived from the L-(alpha 1D), Q-(alpha 1A) and omega-conotoxin GVIA sensitive N-type (alpha 1B) Ca2+ channel alpha 1 subunits. The amplified fragments, which are homologous to the region between domain III and IV of known alpha 1 subunits, reveal splice variation in the L- and Q-type alpha 1 subunit of MES23.5 cells. The transcripts of alpha 1 subunits in these cells were quantified by RNAase protection assay. The data show the existence of different Ca2+ channel types in a single cell line and may reflect multiple functions of voltage operated Ca2+ channels during growth, differentiation and transmitter release.

A large scale preparative gel electrophoresis separation of alpha 1 and alpha 2 subunits of the voltage-gated Ca2+ channel from rabbit skeletal muscle.

A large-scale preparative gel electrophoresis method effectively separates individual voltage-gated calcium channel (VGCC) subunits with high resolution, starting with up to 4 mg of rabbit skeletal muscle L-type VGCC complex. Using this method, we separated alpha 1 and alpha 2 subunits of rabbit skeletal muscle VGCC with a high efficiency and with protein recoveries of 83%. The separated alpha 1 and alpha 2 subunits eluted from the gel in a 1:1 molar ratio. The method should be applicable for separating the other VGCC subunits or subunits of other protein complexes.

Cytotoxicity of immunoglobulins from amyotrophic lateral sclerosis patients on a hybrid motoneuron cell line.

Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs) and inhibit L-type calcium current. To determine whether interaction of ALS IgGs with neuronal VGCCs might influence motoneuron survival, we used a motoneuron-neuroblastoma hybrid (VSC 4.1) cell line expressing binding sites for inhibitors of L-, N-, and P-type VGCCs. Using direct viable cell counts, quantitation of propidium iodide- and fluorescein diacetate-labeled cells, and lactate dehydrogenase release to assess cell survival, we document that ALS IgG kills 40-70% of cAMP-differentiated VSC 4.1 cells within 2 days. ALS IgG-mediated cytotoxicity is dependent on extracellular calcium and is prevented by peptide antagonists of N- or P-type VGCCs but not by dihydropyridine modulators of L-type VGCCs. Preincubating IgG with purified intact L-type VGCC or with isolated VGCC alpha 1 subunit also blocks ALS IgG-mediated cytotoxicity. These results suggest that ALS IgG may directly lead to motoneuron cell death by a mechanism requiring extracellular calcium and mediated by neuronal-type calcium channels.

Amyotrophic lateral sclerosis patient antibodies label Ca2+ channel alpha 1 subunit.

Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord and brain motor neurons. Prior studies demonstrated that most patients with amyotrophic lateral sclerosis possess immunoglobulins that bind to purified L-type voltage-gated calcium channels, that titers of anti-voltage-gated calcium channel antibodies correlate with disease progression rates, and that amyotrophic lateral sclerosis patient-derived antibodies (ALS IgG) produce electrophysiological changes in the function of voltage-gated calcium channels. Using Western transfer immunoblots and enzyme-linked immunosorbent assays, the calcium ionophore-forming alpha 1 subunit of the voltage-gated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS IgG binds. Additionally, the binding of an L-type voltage-gated calcium channel alpha 1 subunit-directed monoclonal antibody, which itself mimics the effects of ALS IgG on skeletal muscle voltage-gated calcium channel currents, is selectively prevented by preaddition of ALS IgG. Voltage-gated calcium channel-binding IgG from patients with Lambert-Eaton myasthenic syndrome appears to be differentiated from ALS IgG by the reactivity of the former to both alpha 1 and beta subunits of the calcium channel. These assays provide further evidence linking amyotrophic lateral sclerosis to an autoimmune process, and suggest one means to differentiate immunoglobulins from patients with amyotrophic lateral sclerosis from those of patients with another autoimmune disease expressing calcium channel antibodies.

ATP-dependent DNA aggregation is a novel function of rat serum albumin.

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.

Differential effect of phosphonoacetic acid on the expression of Epstein-Barr viral antigens and virus production.

The effects of phosphonoacetic acid on cell growth, expression of Epstein-Barr virus antigens, and virus production in human and marmoset lymphoblastoid cell lines have been studied. The drug had no significant effect at concentrations up to 100 mug/ml on cell growth or total cell DNA synthesis. Higher doses induced not only a drastic decrease in DNA synthesis and cell grwoth, but also a dramatic cell enlargement. Immunofluorescence studies showed that greater than or equal to 30 mug/ml of phosphonoacetic acid inhibited viral capsid antigen synthesis without affecting the expression of the nuclear antigen or the spontaneous and 5-iodo-2'-deoxyuridine-induced early antigens. Production of transforming Epstein-Barr virus was also blocked.