DNA amplification of the helper-dependent parvovirus AAV (adeno-associated virus) can be induced by a variety of genotoxic agents in the absence of coinfecting helper virus. Here we investigated whether the origin of AAV type 2 DNA replication cloned into a plasmid is sufficient to promote replication activity in cells treated by the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A pUC19-based plasmid, designated pA2Y1, which contains the left terminal repeat sequences (TRs) representing the AAV origin of replication and the p5 and p19 promoter but lacks any functional parvoviral genes is shown to confer replication activity and to allow selective DNA amplification in carcinogen-treated cells. Following transfection of plasmid pA2Y1 or plasmid pUC19 as a control, density labeling by a bromodeoxyuridine and DpnI resistance assay suggested a semiconservative mode of replication of the AAV origin-containing plasmid. Furthermore, the amount ofDpnI-resistant full-length pA2Y1 DNA molecules was increased by MNNG treatment of cells in a dose-dependent manner. In addition, DNA synthesis of plasmid pA2Y1 was studied in vitro. Extracts derived from MNNG-treated CHO-9 and L1210 cells displayed greater synthesis of DpnI-resistant full-length pA2Y1 molecules than did nontreated controls. Experiments with specific enzyme inhibitors suggested that the reaction is largely dependent on DNA polymerase a, DNA primase, and DNA topoisomerase I. Furthermore, restriction endonuclease mapping analysis of the in vitro reaction products revealed the occurrence of specific initiation at the AAV origin of DNA replication. Though elongation was not very extensive, extracts from carcinogen-treated cells markedly amplified the AAV origin region. Our results, including electron microscopic examination, suggest that the AAV origin/terminal repeat structure is recognized by the cellular DNA replicative machinery induced or modulated by carcinogen treatment in the absence of parvoviral gene products.
We studied the effects of helper-dependent parvovirus AAV [adeno-associated virus] type 2 on carcinogen-inducible resistance to methotrexate (MTX) and adriamycin (ADR) in Chinese hamster ovary cells. Both types of drug resistance were monitored by determination of the number of drug-resistant colonies normalized for the respective value of plating efficiency under non-selective conditions. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) drastically enhanced the frequency of resistance to MTX and ADR. By contrast, infection of cells with AAV-2 prior to treatment with MNNG markedly inhibited carcinogen-induced drug resistance. Infection by AAV alone did not exert any effect. Analysis of the dihydrofolate reductase (dhfr) gene copy numbers of individual MTX-resistant clones derived from MNNG-treated and non-treated cultures revealed similar frequencies (60-80%) and amplitudes of dhfr gene amplification (2- to 8-fold) irrespective of prior AAV treatment. Hence, carcinogen-induced enhancement of MTX-resistance could reflect an increase in the frequency of dhfr gene amplification among the survivors of MNNG treatment. On the other hand, inhibition of carcinogen-inducible drug resistance by AAV suggests an interference of the virus with cellular responses to genotoxic stress, thus leading to enhanced cell killing under altered growth conditions. Possible mechanisms responsible for the inhibitory effect of AAV and its relevance in relation to tumor chemotherapy are discussed.
The rat vascular smooth muscle cell (VSMC) line A10 (ATCC CRL 1476) was stably transfected with a human c-fos promoter-driven luciferase reporter gene to monitor thrombin receptor activation and subsequent induction of c-fos expression. Selective activation of the endogeneous thrombin receptor by the thrombin receptor activating peptide (TRAP1-6), SFLLRN, is shown here to result in a significant transient increase of intracellular [Ca2+], dose-dependent induction of c-fos promoter-mediated luciferase activity, and stimulation of DNA synthesis. These data demonstrate that A10 cells and reporter line derivatives thereof possess a functional thrombin receptor very similar or identical to that previously described. Results obtained with various signal transduction modulating or inhibiting agents support previous notions showing that thrombin receptor activation by SFLLRN is coupled to events involving p21ras activation, protein tyrosine kinase, and activation of PKC. The A10 reporter line described here proved to be a helpful and reliable tool to study alpha-thrombin and TRAP1-6-mediated intracellular events, since it retained most of the spectrum of biological responses found in primary VSMC cultures.
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