Production of mouse models of inherited neurodegenerative diseases is an important step towards understanding the mechanism of neurotoxicity and for testing potential therapies. We are interested in creating a mouse model for X-linked spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by expansion of a CAG repeat within the androgen receptor (AR) gene. To permit generation of mice that will show a SBMA phenotype within their life span, we decided to obtain a yeast artificial chromosome (YAC) carrying the AR gene and introduce CAG repeat mutations numbering 100 or more triplets. SBMA patients with more than 70 CAGs have never been observed; therefore, we chose to expand a 59 CAG repeat tract in vivo in Escherichia coli. Although we set out to expand this repeat tract using a recombination paradigm involving two plasmid co-propagation, we did not observe large expansions. We were instead able to incrementally generate repeat tracts from 100 to 200 CAGs in a yeast integrating plasmid vector by taking advantage of replication instability. In the course of our experiments that yielded these CAG repeat tracts, we evaluated the role of repeat orientation, vector co-propagation, and recA function on the expansion process. We then used one of the yeast integrating vectors to successfully produce an AR YAC construct carrying 100 CAG repeats. AR YAC CAG100 will serve as a valuable reagent for the production of a SBMA mouse.

Two types of transgenic mice were generated to evaluate the role of hydrogen peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes. The other set overexpresses a human catalase construct that is targeted to the nucleus. Expression of the wild-type human catalase transgene was found in liver, kidney, skeletal muscle, heart, spleen, and brain with muscle and heart exhibiting the highest levels. Animals containing the nuclear-targeted construct had a similar pattern of expression with the highest levels in muscle and heart, but with lower levels in liver and spleen. In these animals, immunofluorescence detected catalase present in the nuclei of kidney, muscle, heart, and brain. Both types of transgenic animals had significant increases of catalase activities compared to littermate controls in most tissues examined. Despite enhanced activities of catalase, and its presence in the nucleus, there were no changes in levels of 8OHdG, a marker of oxidative damage to DNA. Nor were there differences in mutant frequencies at a Lac Z reporter transgene. This result suggests that in vivo levels of H2O2 may not generate 8OHdG or other types of DNA damage. Alternatively, antioxidant defenses may be optimized such that additional catalase is unable to further protect nuclear DNA against oxidative damage.

Animal cells generate hydrogen peroxide as a byproduct of energy metabolism. In the presence of reduced metals H(2)O(2) can decompose to a highly reactive hydroxyl radical that attacks essentially all organic molecules, including DNA. We wished to determine if overexpression of catalase and/or the targeting of the enzyme to the nucleus could protect cells from oxidative stress and reduce the frequency of mutation. Wild-type human catalase, which localizes to peroxisomes, and a modified construct, which targets catalase to the nucleus, were overexpressed in a murine line of embryonic carcinoma cells (P19). Both constructs enhanced the resistance of the cells to hydrogen peroxide, but sensitized them to bleomycin. Overexpression of wild-type catalase protected cells against paraquat, while nuclear targeting sensitized them to this agent. Expression of neither construct significantly altered spontaneous mutant frequencies at the endogenous murine adenosine phosphoribosyl transferase (APRT) locus; however, nuclear-targeted catalase prevented an increase in mutant frequency after H(2)O(2) treatment. These results suggest that endogenous levels of hydrogen peroxide may not generate DNA damage in vivo, or that such damage may be efficiently repaired in murine embryonic carcinoma cells.

Ultraviolet light irradiation of DNA in vitro and in vivo induces cyclobutane dimers, (6–4) pyrimidine‐pyrimi‐done photoproducts and a variety of minor products. Using a denned DNA fragment, we have identified two classes of sites that can be cleaved by Escherichia coli endonuclease III: single cytokines whose heat lability corresponds to that of cytosine hydrates and more heat‐stable dipyrimidines containing cytosine. The dipyrimidine products are induced at sites suggestive of (6–4) photoproducts but are not recognized as (6–4) photoproducts by radioimmunoassay. Use of oligonucleotides containing a single cyclobutane thymine dimer, a (6–4) photoproduct or the Dewar photoisomer of the (6–4) photoproduct also indicated that these products are not substrates for endonuclease III. We have therefore identified a minor UV photoproduct that has the same sequence specificity as the two major dipyrimidine photoproducts; it may be a minor isomer, a unique derivative or an oxidative lesion confined to dipyrimidine sites. Its biological significance is not yet known but may be masked by the preponderance of major products at the same sites. Its occurrence at the particular site in dipyrimidine sequences involved in the mutagenic action of UV photoproducts suggests that it may play a role in generating C to T transitions that are common UV‐induced mutations.

Increasing evidence suggests that proteases and their inhibitors play an important role in the etiology of beta-amyloidogenesis and Alzheimer's disease (AD). It is not clear, however, which proteases and protease inhibitors are responsible for the amyloidogenic proteolysis. Candidates include alpha-1-antichymotrypsin, inter-alpha-trypsin inhibitor, and forms of beta-amyloid precursor protein (beta PP) bearing Kunitz protease inhibitor domains. As one approach to this question, we have determined the trypsin inhibitor activity of fibroblast-like cells from 10 familial AD subjects and 20 controls. The activity was quantitated by measuring remaining trypsin activity of reaction mixtures containing trypsin and cell lysates using a fluorogenic substrate and two physiologically distinct populations of fibroblasts: proliferating cells (grown in the presence of 16% serum) and quiescent cells (maintained in 0.1% serum). The remaining trypsin activities of crude protein extracts from proliferating and quiescent AD cultures were not significantly different from those of controls. Perhaps of more general interest to the biology of aging, however, was our finding that protease inhibitor activity increased with the age of the donor (p = 0.005).

The beta amyloid peptide which accumulates within the brains of patients with Alzheimer's disease (AD) is proteolytically derived from a precursor protein (beta PP). We established and characterized four stably transformed human neuroblastoma cell lines which conditionally expressed a partial beta PP fusion protein (amino-17 residues + carboxyl-99 residues; S beta C). Conditional expression of S beta C was achieved using a tetracycline-responsive promoter system. Expression of this fusion protein in one of the cell lines resulted in pronounced cytotoxicity. Addition of n(6),O-2'-dibutyryl adenosine 3',5'-cyclic monophosphate and/or fetal bovine serum to the culture medium of this cell line further elevated the level of S beta C expression and enhanced the associated cytotoxicity. Conditioned medium, acquired from cells expressing S beta C, was not cytotoxic. These findings suggest that modulation of beta PP expression and/or metabolism can have cytotoxic consequences. This is the first report of cytotoxic effects mediated by conditional expression of a beta PP derivative. This immortal cell line provides a unique opportunity to screen for complementary DNAs which suppress this toxicity. Such cDNAs could help elucidate the processes underlying S beta C mediated cytotoxicity which in turn could further our understanding of the pathogenesis of AD and could also provide additional candidate genes for various forms of familial AD.

The primary component of amyloid deposits found in the brains of patients with Alzheimer's disease is the beta-amyloid protein, a derivative of a much larger precursor protein (betaPP). We have previously reported that overexpression of carboxyl (COOH)-terminal fragments of betaPP from an integrated DNA construct leads to degeneration of neuronally differentiating mouse embryonic stem cells and that the neuronal degeneration is related to approximately 14- and 15-kDa COOH-terminal fragments of the precursor protein. We here demonstrate that these putative cytotoxic fragments contain intact beta-amyloid protein. When such transformed cell lines are treated with dimethyl sulfoxide to induce differentiation into muscle cells, however, the resulting muscle cells remain viable (as do control non-transformed cells), despite the production of comparable amounts of the 14- and 15-kDa fragments. These results are consistent with the hypothesis that particular COOH-terminal fragments of betaPP are amyloidogenic and neurotoxic.

The neuropathology of Alzheimer's disease is characterized by the deposition of abnormal protein aggregates. The main constituent of the deposition is beta-amyloid protein. A seminal role of this protein is supported by the discovery of point mutations in the gene of its precursor protein in certain forms of familial Alzheimer's disease. In vitro (cultured neuronal cells), overexpression of the precursor protein or a part of the precursor leads to degeneration of neurons, suggesting neurotoxicity of its derivatives. At this time, all of the reported transgenic mice bearing DNA construct for the precursor or a part of the precursor, however, have not developed convincing pathological changes similar to what is observed in patients with Alzheimer's disease. This interesting discrepancy between in vitro and in vivo suggests suppressors in vivo which ameliorate beta-amyloid precursor protein derivative-mediated neurotoxicity.

Six independent clonal isolates from a morphologically heterogeneous human neuroblastoma cell line stably expressed several products of the human amyloid precursor protein (APP) from an introduced DNA construct; the “substrate-adherent” phenotype (fibroblast-like cells) predominated in all 6; these displayed immunoreactivity of vimentin, but little to no reactivity of neuron-specific enolase. A stably transfected isolate which did not show any expression from the identical construct (presumably because of a position effect) exhibited the predominantly neuronal phenotype of the parental cells (neuron-specific enolase positive). These results suggest selective neurotoxicity of the expressed products. Two of the 6 stably expressing cell lines showed a decrease of native mRNA for APP to levels that were 14-13 that of the parental cells and a decrease of their growth rates to half that of the parental cells; these decreased growth rates were improved by conditioned medium from the parental cell line. Western blot analysis revealed at least four distinct fragments of the COOH-terminus of APP in the isolate which expressed protein and mRNA in greatest abundance, suggesting that overexpression of APP in a human neural cell line leads to aberrant cleavage of APP.

A determination of the mechanisms of drug resistance in tumour cells is important for developing strategies to combat such resistance in persons receiving chemotherapy. This report describes a combined cellular, biochemical, and molecular analysis of a dog kidney cell line selected for resistance to increasing levels of the hydrophilic antifolate, aminopterin. Three distinct drug resistance phenotypes were observed in cells exhibiting high levels of aminopterin resistance. Two of these phenotypes were decreased aminopterin accumulation and increased levels of dihydrofolate reductase specific activity. The third drug resistance phenotype was noted initially as cross resistance to a variety of hydrophobic drugs indicating multidrug resistance. Biochemical assays demonstrated reduced accumulation of the hydrophobic fluorescent drug daunorubicin and of 3H-colchicine in the aminopterin resistant cells. These results were then correlated with increased levels of the multidrug resistance (mdr) gene product, P-glycoprotein, and mdr mRNA levels in the aminopterin resistant cells. However, experiments designed to prove a role for expression of the mdr gene in providing a degree of aminopterin resistance were unsuccessful. It is concluded that aminopterin selection in these dog kidney cells resulted in expression of at least three distinct drug resistance phenotypes and that one of these phenotypes, multidrug resistance, represented a secondary response to the aminopterin selection.

We wished to determine if a partial methylation profile for a specific CpG site was stably maintained in both mammalian tissues and cultured cell lines. To accomplish this, we identified a CpG site with a partial methylation profile located upstream of the mouse adenine phosphoribosyltransferase promoter region. This site was found to be methylated at a level of approximately 25% in mouse brain, kidney, lung, and skeletal muscle tissues, at a level close to 50% in liver, and at level close to 0% in testis. These tissue-specific methylation profiles were not altered during aging. A methylation profile of approximately 25% at this CpG site was also observed in five mouse teratocarcinoma stem cell lines and one additional cultured cell line. This profile, however, was altered upon cellular differentiation, adenine phosphoribosyltransferase hemizygosity, and a loss of adenine phosphoribosyltransferase activity in some of the cultured cell lines. We conclude that partial methylation of a specific CpG site can be stably maintained both in vivo and in vitro and that a mechanism exists for its maintenance. The functional significance of a partial methylation profile remains to be determined.

Three multipotent mouse teratocarcinoma stem lines, all exhibiting unstable expression for the purine salvage enzyme adenine phosphoribosyltransferase (APRT) were used for the isolation of differentiated cell lines from neoplasms developed in syngeneic mice. Two of the stem cell lines (DAP1B and DAP1C) exhibited homozygous deficiencies for APRT expression while the third stem cell line (E140) exhibited a heterozygous deficiency (Turker, M.S., Smith, A.C., and Martin, G.M.; Somat. Cell Mol. Genet.; 10:55-69; 1984). A total of 16 morphologically differentiated cell lines were established from these neoplasms; most were no longer tumorigenic. Differentiated cell lines derived from the E140-induced tumors segregated homozygous deficient mutants in a single step, consistent with their retention of the heterozygous deficient state. Differentiated homozygous deficient cell lines gave rise to phenotypic revertants at very high frequencies (10(-1) to 10(-2)). The majority of these putative revertants, however, yielded cell-free extracts with little or no detectable APRT activity. These putative revertants were capable of adenine salvage and were therefore termed APRT pseudorevertants. Since the APRT pseudorevertant phenotype was only observed in the differentiated progeny of the APRT deficient stem cell lines, we conclude that this change in the nature of the revertant phenotype was a consequence of cellular differentiation.

Chromosomal aberrations were scored in the first metaphases of cells isolated from the kidneys of young (8 months old) and aged (40 months old) CB6F1 mice. There was a sixfold increase in the frequency of such aberrations in metaphases from old mice as compared with young mice.