Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs) MATERIALS AND METHODSCell Culture. Parental embryonic day 15 rat ventral spinal neuron preparation, mouse N18TG2 neuroblastoma culture, somatic cell fusion, and hybrid selection were performed as described (25,26). VSC 4.1 hybrid cells were maintained in logarithmic-phase growth on poly(L-ornithine)-precoated T-75 flasks (Coming) in Dulbecco's modified Eagle's medium/F-12 growth medium (GIBCO) containing Sato's components (Sigma) and 2% heat inactivated newborn calf serum (HyClone). VSC 4.1 cells undergoing cAMP-induced differentiation were plated at 1.2-1.5 x 106 cells per 75-mm2 flask and treated for 7 days with 1 mM dibutyryl cAMP or 1 mM 8-bromo-cAMP before use. Aphidicolin (0.4 pg/ml) (27) (Sigma) was added for 2 days after 24 hr in cAMP, and after 2 additional days of aphidicolin-free growth surviving cells were maintained in medium containing 1 mM cAMP and 0.025-0.05 ,ug of aphidicolin per ml. Differentiated hybrids were harvested by trituration after 1-hr incubation at 370C in Ca2+/Mg2+-free Hanks' balanced salt solution containing 1 mM EDTA and transferred into 24-well plates (Coming) at 2.0 x 104 cells per well [for direct cell count and lactate dehydrogenase (LDH) assays] or per 11-mm-diameter glass coverslip (for fluorescence assays). Pharmacologic agents and immunoglobulins were added to cells 24 hr after replating and were maintained in growth medium for the entire experAbbreviations: ALS, amyotrophic lateral sclerosis; LEMS, Lam-bert-Eaton myasthenic syndrome; VGCC, voltage-gated calcium channel; FDA, fluorescein diacetate; PI, propidium iodide; LDH, lactate dehydrogenase; AMPA, DL-a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione.tPresent address:

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 receptorexpressing cell line (RKO) indicated that Sp1, Sp3, and a factor similar to, but distinct from, activator protein-2␣ bound to this region. Mutations preventing the binding of the activator protein 2␣-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-2␣A expression. Although using GEO nuclear extract, little activator protein-2␣-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-2␣-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-2␣-related factor.

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.

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 TNFa or other apoptotic agents. FACS analysis showed that 5a interacted synergistically with ligands of the TNF receptor superfamily, including TNFa and TNF receptor-like apoptosisinducing 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 TNFa. Compound 5a did not act directly on the mitochondrion or via changes in protein synthesis. Instead, the mechanism requires ligandreceptor 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.

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