Proceedings of the National Academy of Sciences of the United States of America, May 1, 1976
The effects of phosphonoacetic acid on cell growth, expression of Epstein-Barr virus antigens, an... more 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 jg/ml on cell growth or total cell DNA synthesis. Hi' er doses induced not only a drastic decrease in DNA synthesis and cell growth, but also a dramatic cell enlargement. Immunofluorescence studies showed that >30 jsg/ml of phosphonoacetic acid inhibited viral capsid antigen synthesis without affecting the expression of the nuclear antigen or the spontaneous and 5-iodo-2'-deoxyuridineinduced early antigens. Production of transforming Epstein-Barr virus was also blocked. Inhibition of viral capsid antigen expression and of virus production at low doses was reversible. The transforming virus produced after the removal
Proceedings of the National Academy of Sciences of the United States of America, Apr 12, 1994
Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type ske... more 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 iodideand fluorescein diacetate-labeled cells, and lactate dehydrogenase release to assess cell survival, we document that ALS IgG kills 40-70% of cAMPdifferentiated VSC 4.1 cells within 2 days. ALS IgG-mediated cytotoxicity is dependent on extracellular calcium and is prevented by peptide antagonists of Nor P-type VGCCs but not by dihydropyridine modulators of L-type VGCCs. Preincubating IgG with purified intact L-type VGCC or with isolated VGCC al 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 (ALS) is a catastrophic human neurodegenerative syndrome that compromises upper and lower motoneuron function, leading to progressive paralysis and death. While pathogenesis of ALS is unknown, recent research has provided significant clues toward understanding the causes of selective neuronal death in this syndrome. Familial ALS (which affects 10% of ALS patients) is linked in some kindreds to mutations in Cu2+/Zn2+-dependent superoxide dismutase, thereby suggesting the potential role of free radicals in motoneuron injury (1). In patients with sporadic ALS, motoneuron death is hypothesized to result
Several studies have shown that matrix metalloproteases (MMPs) promote tumor growth, invasion, an... more 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.
Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord... more 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 (x subunit of the voltagegated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS I g G binds. Additionally, the binding of an L-type voltage-gated calcium channel (xL subunit-directed monoclonal antibody, which itself mimics the effects of ALS I g G 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 0 1~ and p 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.
Sporadic amyotrophic lateral sclerosis is a motor neuron disease of unknown origin. Autoimmunity ... more Sporadic amyotrophic lateral sclerosis is a motor neuron disease of unknown origin. Autoimmunity against voltagegated 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 IX I 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 a1 subunit by diethylaminoethyl cellulose (DEAE)-purified amyotrophic lateral sclerosis IgG was not observed with Fab fragments of amyotrophic lateral sclerosis I&. 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. Nyormoi 0. Proreolytic activity in dmyotrophic l'iteral sclerosis IgG preprations. Anti Neurol 1996;40:70 1-706 Amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease is a human disease in which the upper and lower niotor neurons degenerate, leading to paralysis and death [I]. The disease consists of two subrypes: familial and sporadic. The former comprises about
A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension cult... more A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension culture of an established Burkitt lymphoma cell line (Raji). Except for the altered morphology associated with substrate attachment, Raji-A is identical to Raji with respect to karyotype, isozyme composition. susceutibilitv to Epstein-Barr virus (EBV) infection and inducibilitv of latent EBV. In order to facilitate fusion experiments with RajiiA, drug resistant variants were induced by treating cells with ethylmethane sulfonate followed by selection in growth medium which contains 8azaguanine. Three clones (AGRO, AGR3 and AGR6) were found to be resistant to 5O,ug/ml of 8-azaguanine. They had only 10-15 % as much hypoxanthineguanine phosphoribosyltransferase activity as wild type cells and a low plating efficiency of l-3 x 1OW in the selective medium, HAT. Potential uses of these variants for studying EBV-lymphoblastoid cell interactions as well as human genetics by cell hybridization are discussed.
EBV was found to be activated by cocultivation of EBV transformed lymphoblastoid cell ,!ines and ... more EBV was found to be activated by cocultivation of EBV transformed lymphoblastoid cell ,!ines and a mammary carcinoma cell line, MCF-7. Both non-EB V producer and producer cell lines are susceptible to this activation. However not every EBV-transformed cell line can be activated. Although the mechanism oJ activation was investigated in a number of ways, it is still unknown whether the process requires cell to cell contact, if the ir, ducing agent is secreted in the medium and where in the host cell or the virus cycle it act~,.
Proliferating hybrids were formed between rat spermatids and LM(TK~) cl 1 D, an established line ... more Proliferating hybrids were formed between rat spermatids and LM(TK~) cl 1 D, an established line of mouse fibroblasts. Spermatids were obtained from dissociated rat seminiferous tubules by differential attachment of testicular somatic cells to culture plates, followed by gravity velocity sedimentation into linear Ficoll gradients. Cells were fused with the aid of Sendai virus and hybrid selection was facilitated by the inability of cl 1 D cells to grow in HAT medium and the inability of spermatids to attach or proliferate in vitro. Three types of cells grew in fusion plates: (1) unfused contaminant rat testicular somatic cells; (2) rat testicular cell x cl 1 D hybrids and (3) rat spermatid x cl 1 D hybrids. Unfused rat testicular somatic cells were morphologically and karyologically different from either type of hybrid. The 2 hybrid classes were distinguished by the total number of chromosomes and the number of parental chromosome markers. Rat chromosomes are preferentially eliminated from haploid as well as diploid hybrids and at least 3 rat enzymes (lactate dehydrogenase, glucose phosphate isomerase and an unknown oxidase) are synthesized in hybrid clones of both types. The implications of partial haploid hybrids for genetic analysis are briefly discussed.
This study shows a strong association between cell attachment to substratum and activation of β1-... more This study shows a strong association between cell attachment to substratum and activation of β1-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 anchoragedependent 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 µM) or CPT-11 (40 µM) 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 β1-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.
ABSTRACT Studies of cell injury and death in Alzheimer's disease have suggested a promine... more ABSTRACT Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β-amyloid peptide (β-AP), a 40–43-amino-acid peptide derived from a larger membrane glycoprotein, β-amyloid precursor protein (β-APP). Previous experiments have demonstrated that β-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β-APP mRNA content is increased 3.5-fold in 24 h after treatment with β-AP1–40. Accompanying β-AP1–40-induced cell injury, levels of cell-associated β-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of β-APP and 12- and 4-kDa fragments are also increased. Application of β-APP antisense oligodeoxynucleotide reduces both cytotoxicity and β-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β-APP expression. These results suggest a selective positive feedback mechanism whereby β-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β-APP with the net consequence of further neuronal damage.
A large scale preparative gel electrophoresis separation of a, and a2 subunits of the voltage-gat... more A large scale preparative gel electrophoresis separation of a, and a2 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 a, and a, subunits of rabbit skeletal muscle VGCC with a high efficiency and with protein recoveries of 83%. The separated a, and a, subunits eluted from the gel in a 1:l molar ratio. The method should be applicable for separating the other VGCC subunits or subunits of other protein complexes.
The sensitivity of Burkitt lymphoma-derived cell lines to Epstein-Barr viruy (EBV) superinfection... more The sensitivity of Burkitt lymphoma-derived cell lines to Epstein-Barr viruy (EBV) superinfection and the inducibility of the latent EBVgenome in these lines were studied by somatic cell hybridization. Two non-EBV producer lines: ACR3, an adherent, 8azaguanine-resistant variant of the Rajiline; and Namalwa, a non-adherent, 8-azaguaninesensitive line, were fused with the aid of P-propiolactone-inactivated Sendai virus. The resident EB V genome in Raji is inducible with 5-iododeoxyuridine and the line is also sensitive to EBV superinfection. Namalwa is relatively resistant to both super-infec tion and induction, and synthetizes surface-associated IgM-lambda. Cytological studies showed that hybrid cells appear to be much larger than either parent and attach to culture plates less firmly than the adherent Raji variant parent. Karyological ana1yse.c showed that hybrids contain the expected sum of the parental chromosome markers. Membrane immunofluorescence tests also showed that hybrids synthetize IgM. All the hybrid cells appear to be non-EBVproducers, but they are sensitive to both EBV superinfection and induction of latent EB V. These findings suggest the following explanations. (I) there is no evidence of any complementation between the two non-EBVproducer lines (Raji and Namalwa) to elicit spontaneous EB V production in hybrid cells; (2) Namalwa is deficient in some factors required for the synthesis of EBV-specified eurly antigens after EBV superinfection and after induction of latent EBV by IUdR: these factors are supplied by the Rajiparerit in the hybrids; or (3) Raji, Namalwa and hybrid cells or EBV all produce a substance which inhibits activation of a productive viral cycle, but whose action is antagonized in Raji and hybrid cells to allow the synthesis of EBVspecific early antigens. The mechanism which regulates the functional indefinitely in non-EBV produccr cell lincs and and physical relationship between EBV and in a latent state with occasional spontaneous human lyrnphoblastoid cells is a n intriguing activation of a productive cycle in a minority of the phenomenon which has yet to be elucidated. cells in producer cell lines (Hinuma and Grace, Nevertheless, this mechanism appears to have 1967; Maurer et al., 1970; Klein et al., 1972). several levels of action. First, it can maintain EBV Second, it regulates thc rensitivity of human
Proliferating hybrids were formed between rat spermatids and LM(TK-)cl 1 D, an established line o... more Proliferating hybrids were formed between rat spermatids and LM(TK-)cl 1 D, an established line of mouse fibroblasts. Spermatids were obtained from dissociated rat seminiferous tubules by differential attachment of testicular somatic cells to culture plates, followed by gravity velocity sedimentation into linear Ficoll gradients. Cells were fused with the aid of Sendai virus and hybrid selection was facilitated by the inability of cl 1 D cells to grow in HAT medium and the inability of spermatids to attach or proliferate in vitro. Three types of cells grew in fusion plates: (1) unfused contaminant rat testicular somatic cells; (2) rat testicular cell x cl 1 D hybrids and (3) rat spermatid x cl 1 D hybrids. Unfused rat testicular somatic cells were morphologically and karyologically different from either type of hybrid. The 2 hybrid classes were distinguished by the total number of chromosomes and the number of parental chromosome markers. Rat chromosomes are preferentially eliminate...
Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for b... more 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 f...
Proceedings of the National Academy of Sciences of the United States of America, May 1, 1976
The effects of phosphonoacetic acid on cell growth, expression of Epstein-Barr virus antigens, an... more 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 jg/ml on cell growth or total cell DNA synthesis. Hi' er doses induced not only a drastic decrease in DNA synthesis and cell growth, but also a dramatic cell enlargement. Immunofluorescence studies showed that >30 jsg/ml of phosphonoacetic acid inhibited viral capsid antigen synthesis without affecting the expression of the nuclear antigen or the spontaneous and 5-iodo-2'-deoxyuridineinduced early antigens. Production of transforming Epstein-Barr virus was also blocked. Inhibition of viral capsid antigen expression and of virus production at low doses was reversible. The transforming virus produced after the removal
Proceedings of the National Academy of Sciences of the United States of America, Apr 12, 1994
Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type ske... more 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 iodideand fluorescein diacetate-labeled cells, and lactate dehydrogenase release to assess cell survival, we document that ALS IgG kills 40-70% of cAMPdifferentiated VSC 4.1 cells within 2 days. ALS IgG-mediated cytotoxicity is dependent on extracellular calcium and is prevented by peptide antagonists of Nor P-type VGCCs but not by dihydropyridine modulators of L-type VGCCs. Preincubating IgG with purified intact L-type VGCC or with isolated VGCC al 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 (ALS) is a catastrophic human neurodegenerative syndrome that compromises upper and lower motoneuron function, leading to progressive paralysis and death. While pathogenesis of ALS is unknown, recent research has provided significant clues toward understanding the causes of selective neuronal death in this syndrome. Familial ALS (which affects 10% of ALS patients) is linked in some kindreds to mutations in Cu2+/Zn2+-dependent superoxide dismutase, thereby suggesting the potential role of free radicals in motoneuron injury (1). In patients with sporadic ALS, motoneuron death is hypothesized to result
Several studies have shown that matrix metalloproteases (MMPs) promote tumor growth, invasion, an... more 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.
Sporadic amyotrophic lateral sclerosis is an idiopathic human degenerative disease of spinal cord... more 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 (x subunit of the voltagegated calcium channel is now identified as the major voltage-gated calcium channel antigen to which ALS I g G binds. Additionally, the binding of an L-type voltage-gated calcium channel (xL subunit-directed monoclonal antibody, which itself mimics the effects of ALS I g G 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 0 1~ and p 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.
Sporadic amyotrophic lateral sclerosis is a motor neuron disease of unknown origin. Autoimmunity ... more Sporadic amyotrophic lateral sclerosis is a motor neuron disease of unknown origin. Autoimmunity against voltagegated 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 IX I 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 a1 subunit by diethylaminoethyl cellulose (DEAE)-purified amyotrophic lateral sclerosis IgG was not observed with Fab fragments of amyotrophic lateral sclerosis I&. 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. Nyormoi 0. Proreolytic activity in dmyotrophic l'iteral sclerosis IgG preprations. Anti Neurol 1996;40:70 1-706 Amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease is a human disease in which the upper and lower niotor neurons degenerate, leading to paralysis and death [I]. The disease consists of two subrypes: familial and sporadic. The former comprises about
A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension cult... more A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension culture of an established Burkitt lymphoma cell line (Raji). Except for the altered morphology associated with substrate attachment, Raji-A is identical to Raji with respect to karyotype, isozyme composition. susceutibilitv to Epstein-Barr virus (EBV) infection and inducibilitv of latent EBV. In order to facilitate fusion experiments with RajiiA, drug resistant variants were induced by treating cells with ethylmethane sulfonate followed by selection in growth medium which contains 8azaguanine. Three clones (AGRO, AGR3 and AGR6) were found to be resistant to 5O,ug/ml of 8-azaguanine. They had only 10-15 % as much hypoxanthineguanine phosphoribosyltransferase activity as wild type cells and a low plating efficiency of l-3 x 1OW in the selective medium, HAT. Potential uses of these variants for studying EBV-lymphoblastoid cell interactions as well as human genetics by cell hybridization are discussed.
EBV was found to be activated by cocultivation of EBV transformed lymphoblastoid cell ,!ines and ... more EBV was found to be activated by cocultivation of EBV transformed lymphoblastoid cell ,!ines and a mammary carcinoma cell line, MCF-7. Both non-EB V producer and producer cell lines are susceptible to this activation. However not every EBV-transformed cell line can be activated. Although the mechanism oJ activation was investigated in a number of ways, it is still unknown whether the process requires cell to cell contact, if the ir, ducing agent is secreted in the medium and where in the host cell or the virus cycle it act~,.
Proliferating hybrids were formed between rat spermatids and LM(TK~) cl 1 D, an established line ... more Proliferating hybrids were formed between rat spermatids and LM(TK~) cl 1 D, an established line of mouse fibroblasts. Spermatids were obtained from dissociated rat seminiferous tubules by differential attachment of testicular somatic cells to culture plates, followed by gravity velocity sedimentation into linear Ficoll gradients. Cells were fused with the aid of Sendai virus and hybrid selection was facilitated by the inability of cl 1 D cells to grow in HAT medium and the inability of spermatids to attach or proliferate in vitro. Three types of cells grew in fusion plates: (1) unfused contaminant rat testicular somatic cells; (2) rat testicular cell x cl 1 D hybrids and (3) rat spermatid x cl 1 D hybrids. Unfused rat testicular somatic cells were morphologically and karyologically different from either type of hybrid. The 2 hybrid classes were distinguished by the total number of chromosomes and the number of parental chromosome markers. Rat chromosomes are preferentially eliminated from haploid as well as diploid hybrids and at least 3 rat enzymes (lactate dehydrogenase, glucose phosphate isomerase and an unknown oxidase) are synthesized in hybrid clones of both types. The implications of partial haploid hybrids for genetic analysis are briefly discussed.
This study shows a strong association between cell attachment to substratum and activation of β1-... more This study shows a strong association between cell attachment to substratum and activation of β1-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 anchoragedependent 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 µM) or CPT-11 (40 µM) 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 β1-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.
ABSTRACT Studies of cell injury and death in Alzheimer's disease have suggested a promine... more ABSTRACT Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β-amyloid peptide (β-AP), a 40–43-amino-acid peptide derived from a larger membrane glycoprotein, β-amyloid precursor protein (β-APP). Previous experiments have demonstrated that β-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β-APP mRNA content is increased 3.5-fold in 24 h after treatment with β-AP1–40. Accompanying β-AP1–40-induced cell injury, levels of cell-associated β-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of β-APP and 12- and 4-kDa fragments are also increased. Application of β-APP antisense oligodeoxynucleotide reduces both cytotoxicity and β-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β-APP expression. These results suggest a selective positive feedback mechanism whereby β-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β-APP with the net consequence of further neuronal damage.
A large scale preparative gel electrophoresis separation of a, and a2 subunits of the voltage-gat... more A large scale preparative gel electrophoresis separation of a, and a2 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 a, and a, subunits of rabbit skeletal muscle VGCC with a high efficiency and with protein recoveries of 83%. The separated a, and a, subunits eluted from the gel in a 1:l molar ratio. The method should be applicable for separating the other VGCC subunits or subunits of other protein complexes.
The sensitivity of Burkitt lymphoma-derived cell lines to Epstein-Barr viruy (EBV) superinfection... more The sensitivity of Burkitt lymphoma-derived cell lines to Epstein-Barr viruy (EBV) superinfection and the inducibility of the latent EBVgenome in these lines were studied by somatic cell hybridization. Two non-EBV producer lines: ACR3, an adherent, 8azaguanine-resistant variant of the Rajiline; and Namalwa, a non-adherent, 8-azaguaninesensitive line, were fused with the aid of P-propiolactone-inactivated Sendai virus. The resident EB V genome in Raji is inducible with 5-iododeoxyuridine and the line is also sensitive to EBV superinfection. Namalwa is relatively resistant to both super-infec tion and induction, and synthetizes surface-associated IgM-lambda. Cytological studies showed that hybrid cells appear to be much larger than either parent and attach to culture plates less firmly than the adherent Raji variant parent. Karyological ana1yse.c showed that hybrids contain the expected sum of the parental chromosome markers. Membrane immunofluorescence tests also showed that hybrids synthetize IgM. All the hybrid cells appear to be non-EBVproducers, but they are sensitive to both EBV superinfection and induction of latent EB V. These findings suggest the following explanations. (I) there is no evidence of any complementation between the two non-EBVproducer lines (Raji and Namalwa) to elicit spontaneous EB V production in hybrid cells; (2) Namalwa is deficient in some factors required for the synthesis of EBV-specified eurly antigens after EBV superinfection and after induction of latent EBV by IUdR: these factors are supplied by the Rajiparerit in the hybrids; or (3) Raji, Namalwa and hybrid cells or EBV all produce a substance which inhibits activation of a productive viral cycle, but whose action is antagonized in Raji and hybrid cells to allow the synthesis of EBVspecific early antigens. The mechanism which regulates the functional indefinitely in non-EBV produccr cell lincs and and physical relationship between EBV and in a latent state with occasional spontaneous human lyrnphoblastoid cells is a n intriguing activation of a productive cycle in a minority of the phenomenon which has yet to be elucidated. cells in producer cell lines (Hinuma and Grace, Nevertheless, this mechanism appears to have 1967; Maurer et al., 1970; Klein et al., 1972). several levels of action. First, it can maintain EBV Second, it regulates thc rensitivity of human
Proliferating hybrids were formed between rat spermatids and LM(TK-)cl 1 D, an established line o... more Proliferating hybrids were formed between rat spermatids and LM(TK-)cl 1 D, an established line of mouse fibroblasts. Spermatids were obtained from dissociated rat seminiferous tubules by differential attachment of testicular somatic cells to culture plates, followed by gravity velocity sedimentation into linear Ficoll gradients. Cells were fused with the aid of Sendai virus and hybrid selection was facilitated by the inability of cl 1 D cells to grow in HAT medium and the inability of spermatids to attach or proliferate in vitro. Three types of cells grew in fusion plates: (1) unfused contaminant rat testicular somatic cells; (2) rat testicular cell x cl 1 D hybrids and (3) rat spermatid x cl 1 D hybrids. Unfused rat testicular somatic cells were morphologically and karyologically different from either type of hybrid. The 2 hybrid classes were distinguished by the total number of chromosomes and the number of parental chromosome markers. Rat chromosomes are preferentially eliminate...
Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for b... more 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 f...
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