ABSTRACT
Immortalized rat fibroblasts, genetically altered to secrete NGF, BDNF, and bFGF, were implanted in rat brain near the striatum 7 days before striatal infusion of excitotoxic quantities of an NMDA-receptor agonist. Analysis of striatal damage 7 days after lesioning revealed that implantation of NGF-secreting cells reduced the size of the excitotoxic lesions by more than 80% when compared with control cells, while implanting of bFGF-secreting cells caused a 30% decrease in excitotoxic lesion size. BDNF-secreting fibroblasts caused no protective sparing in the striatum in this lesion model. This finding shows that biological delivery of NGF and bFGF by grafting of genetically altered cells protects against glutamate toxicity in the adult striatum while grafting of BDNF-producing cells does not. Such observations begin to define a spectrum of neurotrophic agents able to mitigate the cell loss seen in neurodegeneration.
Subject(s)
Corpus Striatum/drug effects , Fibroblast Growth Factor 2/pharmacology , Nerve Degeneration/drug effects , Nerve Growth Factors/pharmacology , Nerve Tissue Proteins/pharmacology , Acetylcholine/physiology , Animals , Brain-Derived Neurotrophic Factor , Cell Line , Disease Models, Animal , Fibroblast Growth Factor 2/metabolism , Fibroblasts/transplantation , Nerve Fibers/chemistry , Nerve Growth Factors/metabolism , Nerve Tissue Proteins/metabolism , Rats , Tyrosine 3-Monooxygenase/analysisABSTRACT
The mutagenic and carcinogenic substance benzo[a]pyrene reacts with DNA following activation to its corresponding 7,8-diol 9,10-epoxide (BPDE), and the major DNA adduct (BP-N2-Gua) is formed when the C(10)-position of BPDE reacts with the N2-position of guanine. It is unknown if this adduct is a premutagenic lesion in vivo. Herein, the construction and characterization of an M13mp19-based, E. coli vector that contains BP-N2-Gua located in the unique PstI restriction endonuclease recognition site at nucleotide position 6249 in the (-)-strand is described (designated, BP-N2-Gua-M13mp19). First, the oligonucleotide 5'-TGCA-3' was reacted with BPDE and a product (5'-T(BP-N2)GCA-3') was isolated by HPLC that, when enzymatically digested to deoxynucleosides, yielded an adduct that comigrated on HPLC with an authentic BP-N2-Gua deoxynucleoside standard. Second, the 5'-hydroxyl group of 5'-T-(BP-N2)GCA-3' was phosphorylated with ATP and T4 polynucleotide kinase, and the product (5'-pT(BP-N2)GCA-3') was purified by HPLC. This product is stable when heated at 80 degrees C at both neutral and alkaline pH. Third, M13mp19 was manipulated such that the sequence 5'-pTGCA-3' was selectively removed from the (-)-strand in its unique PstI recognition site, and 5'-pT(BP-N2)GCA-3' was ligated into this gap with T4 DNA ligase and ATP. The product of this reaction (BP-N2-Gua-M13mp19) was shown to be insensitive to cleavage by PstI, which suggests that a modification is located in the PstI recognition site. The most likely modification is the adduct BP-N2-Gua.
Subject(s)
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide , Benzo(a)pyrene/metabolism , DNA Adducts , DNA , Escherichia coli/genetics , Genetic Vectors , Base Sequence , Biotransformation , Genome, Bacterial , Oligodeoxyribonucleotides/chemical synthesisABSTRACT
Rat pheochromocytoma PC12 cells have been modified genetically by the use of replication-defective retroviral vectors containing either the bacterial gene for beta-galactosidase (lac Z) or cDNAs for mouse beta-nerve growth factor (NGF) and the bacterial gene for neomycin resistance. Using the lac Z vector, clonal lines of PC12 cells were obtained in which almost 100% of cells stably expressed this histochemical marker. Infection of PC12 cells or the derived subclone PC12-BAG, which expresses beta-galactosidase, with the NGF vectors resulted in autocrine differentiation as assessed by extensive neurite formation, which occurred within hours after infection and was maintained for weeks in culture. Neurite formation could be partially blocked by antibodies to NGF. The percentage of cells expressing neurite outgrowth was greater than that of PC12 cells treated with exogenous NGF. PC12 cells infected with the NGF vectors were shown to release this trophic factor into the medium using a two-site enzyme immunoassay and a bioassay on 'naive' PC12 cells. PC12 cells genetically modified using these vectors provide a means to: follow the fate of the cells after transplantation into animals; test for delivery in vivo of NGF and catecholamines by grafted, autocrine-differentiated PC12 cells; and study the long-term actions of NGF on responsive cells without adding exogenous NGF.