Frequency Of Gene Amplification Research Articles

Increase of extrachromosomal circular DNA in mouse 3T6 cells on perturbation of DNA synthesis: Implications for gene amplification

We have analyzed the amount of extrachromosomal double-stranded covalently closed circular nonmitochondrial DNA in mouse 3T6 cells by Southern blotting and electron microscopy. Treatment with 7,1-dimethylbenz[a]anthracene, known to promote amplification of integrated SV40 genomes, elevated the amount of circular DNA. Inhibition of DNA synthesis with hydroxyurea, earlier shown to enhance amplification of the cellular dihydrofolate reductase gene, resulted in yet higher levels. Thus, elevation of the frequency of gene amplification and generation of extrachromosomal circular DNA seem to accompany each other in the situations studied in this paper. Two other DNA synthesis inhibitors, aphidicolin and thymidine, had markedly lesser effects on circular DNA. The significance of these findings for the mechanism of gene amplification is discussed.

Analysis of gene amplification in human tumor cell lines.

Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.

Changes in H1 content, nucleosome repeat lengths and DNA elongation under conditions of hydroxyurea treatment that reportedly facilitate gene amplification

Depletion of histone H1, changes in nucleosome repeat lengths, and extents of DNA elongation were investigated in synchronized Chinese hamster (line CHO) cells using the general conditions of hydroxyurea treatment that appear to increase the frequency of gene amplification, i.e., synchronized cultures of G 1 cells were allowed to begin to enter S phase before treatment with hydroxyurea was effected to retard DNA synthesis (Mariani, B.D. and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901–1910). During the time that synchronized G 1 cells begin to enter S phase, there occur considerable synchrony decay and accumulation of new DNA that increase with time before treatment with hydroxyurea is initiated. During exposure to hydroxyurea, there occur depletion of histone H1 and shortened repeat lengths for the DNA synthesized in the presence of hydroxyurea. In contrast, DNA synthesized in S phase before exposure to hydroxyurea has essentially the same repeat lengths as bulk chromatin at both the time that hydroxyurea treatment is effected and after 6 h in its presence. Sedimentation measurements indicate that the early replicating DNA undergoes considerable elongation both before and during 6 h of exposure to 0.3 mM hydroxyurea. Thus, nearly all of the early replicating DNA is elongated to greater than average replicon size under those conditions of hydroxyurea treatment that appear to favor gene amplification. Because the extents of DNA synthesis and cell cycle progression vary as functions of drug concentration, treatment times, and unknown factors (from experiment to experiment), it would appear that the parameters must be carefully monitored in each experiment if biochemical results are to be related to the position of cells in the growth cycle.

Transient hypoxia enhances the frequency of dihydrofolate reductase gene amplification in Chinese hamster ovary cells.

Exposure of Chinese hamster cells to reduced oxygen partial pressure results in a marked enhancement in the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. The frequency of enhanced resistance is a function of the length of exposure to hypoxic conditions and the time after recovery from hypoxia when cells are plated into methotrexate-containing medium. Hypoxia results in an inhibition of DNA synthesis; upon return to normal oxygen atmosphere, greater than 60% of cells in S phase at the time hypoxia was started subsequently undergo overreplication of DNA within a single cell cycle. The cells with the increased frequency of gene amplification are derived from this subset of overreplicated cells. These results are discussed within the context of the hypoxic state of many solid tumors and the high frequency of aneuploidy, chromosomal aberrations, and spontaneously occurring resistances to a number of cancer chemotherapeutic agents.

Time-dependent changes in H1 content, H1 turnover, DNA elongation, and the survival of cells blocked in early S phase by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine.

Cells were synchronized in G1 by isoleucine deprivation and then released into medium containing 1 mM hydroxyurea (HU), 5 micrograms mL-1 aphidicolin (APC), or 1 microgram mL-1 5-fluorodeoxyuridine (fl5dU). Coulter volume, content of histone H1 per unit DNA, turnover of histone H1, the extent of DNA elongation, and the survival of cells were measured as functions of time after release into the presence of the drugs. At the concentrations used in the experiments, the drug differ in their toxicity (fl5dU greater than HU greater than APC), induction of unbalanced cell growth, and the distribution of new DNA fragment sizes allowed during block, but they all (1) allow cells to enter S phase, (2) cause similar time-dependent losses of histone H1 per unit DNA, which begin as synchronized G1 cells begin to enter S phase, (3) retard DNA elongation beyond replicon size, and (4) retard the turnover of histone H1. The results indicate that loss of histone H1, inhibition of histone turnover, the retarded ligation of newly replicated DNA into bulk chromatin, and chromatin structural changes may be part of the cell's general response to inhibition of DNA replication. Since transient S phase block increases the frequencies of gene amplification [Mariani, B. D., & Schimke, R. T. (1984) J. Biol. Chem. 259, 1901-1910] and sister chromatid exchanges (SCE) [Rainaldi, G., Sessa, M. R., & Mariani, T. (1984) Chromosoma 90, 46-49], the observed changes in H1 content and chromatin organization may also be essential features of gene amplification and SCE.