Gene amplification in methotrexate-resistant mouse cells

The amplification units in human tumors containing amplified myc genes were examined. The amplifilcation unit in all cases consisted of a large genomic region coamplified with the coding region of the myc genes themselves. In eight independent neuroblastomas containing N-myc amplifications, the amplification unit was estimated to be 290 to 430 kilobases. This amplification unit was highly conserved among the different neuroblastomas, with some neuroblastomas containing almost identical units. In contrast, five tumor cell lines containing c-myc amplifications exhibited amplification units that were more variable in size (90 to 300 kilobases) and sequence content; at least three different patterns of c-myc amplification units could be discerned.It has been shown that increased levels of normal cellular oncogene products are capable of transforming cells (1, 2). Gene amplification provides a potent mechanism for increasing the amount of a normal cellular oncogene product (reviewed in refs. [3][4][5]. Amplification of various oncogenes has been reported in a variety of human tumors or cell lines derived from human tumors. These reports have included amplification of c-erbB in brain tumors and a squamous cell carcinoma (6-8), c-abl in leukemia (9, 10), c-myb in colon cancer and leukemia (11,12), N-myc in neuroblastomas and retinoblastomas (13)(14)(15)(16), and c-myc in several types of tumors (17-21). Despite the fact that amplified oncogenes have occurred in this wide variety of neoplasms, little is known about the mechanism of oncogene amplification or the structure of their amplification units.At the chromosomal level, the amplified oncogenes are contained within double minute chromosomes (DM) or homogeneously staining regions (HSR) (reviewed in refs. [3][4][5]. DM and HSR also occur in drug resistant cell lines. In such cases, it has been shown that the genes conferring drug resistance are amplified within a large domain of DNA that includes 100 to 1000 kilobases (kb) of sequences flanking the drug resistance genes themselves (22-26). The amplified gene and coamplified flanking sequences form the amplification unit. An understanding of the structure of the amplification unit is essential to delineating the mechanism of amplification.We have now partially characterized the amplification units in eight human neuroblastomas containing amplified N-myc genes and five human cell lines containing amplified c-myc genes. In both cases, large domains of coamplified sequences accompanied the amplification of the coding region of the oncogene. The N-myc amplification unit was approximately 290 to 430 kb in length and was highly conserved among neuroblastomas from eight different patients. The size of the c-myc amplification unit was much more variable, ranging from 90 to 300 kb. The patterns of amplified sequences indicated that at least three different c-myc amplification units were represented by the five cell lines studied. These results with oncogene amplification units are compared with the amplification units i...

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Amplification units containing human N-myc and c-myc genes.

Kinzler¹,

Zehnbauer

Brodeur

et al. 1986

“…Originally reported by Biedler and Spengler (10) to be present in antifolate-resistant Chinese hamster lung cells overproducing dihydrofolate reductase, as well as in cells of two different neuroblastoma lines, SK-N-BE(2) and IMR-32, HSRs have since been demonstrated by hybridization in situ to contain amplified copies of (i) the DHFR gene in antifolate-resistant mouse and Chinese hamster cells (11,12); (ii) DNA sequences that encode mRNA species whose function is not yet known in the mouse adrenocortoid tumor cell line Y-1 (13); and (iii) the c-myc gene in the neuroendocrine tumor cell line COLO 320 (14). Similarly, a large body of evidence now exists which suggests that DMs contain amplified DNA (13)(14)(15)(16)(17)(18), although direct demonstration by hybridization in situ has not been possible due to their small size and random nonchromosomal location. Indeed, it is most likely that HSRs and DMs are alternate cytological manifestations of the same phenomenon-i.e., the somatic cell amplification of genomic DNA.…”

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confidence: 99%

Specific DNA sequence amplification in human neuroblastoma cells.

Montgomery

Biedler

Spengler

et al. 1983

Southern blot analysis of a number of EcoRI-digested human neuroblastoma DNAs has revealed the presence of a family of discrete restriction fragments, the majority of which are amplified in most, but not all, of the neuroblastoma cell lines tested. None of these sequences is abundantly present in DNA from other human tumors of different tissue origins, including several either known or presumed to contain amplified DNA. Hence, these sequences appear to be specifically amplified by neuroblastoma cells. Hybridization with metaphase chromosomes in situ has localized these sequences to either the homogeneously staining regions or double-minute chromosomes of different neuroblastoma cell lines, indicating that these chromosomal structures, although present in cell lines established from different patients, share many sequences and may have a common, but as yet unknown, function.

“…Other processes shown to be altered by local mutation over a large fraction of the genome are sporulation in Bacillus subtilis (38), penicillin production by Aspergillus nidulans (39) and the development of the several organs in Drosophila melanogaster (40). In addition, extensive DNA rearrangement is found after exposure to MTX, and the DNA sequences seem to differ in each cell line (30). They include nondihydrofolate reductase as well as dihydrofolate reductase sequences.…”

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confidence: 99%

“…Such effects as myeloid suppression, gastrointestinal symptoms, hepatotoxicity, alopecia, and osteopathy might be perpetuated in chronic form long after drug administration as a result of subnormal performance of heritably damaged cells. The well-known occurrence of chromosome aberrations in MTX-treated cells (14,30) might explain the occurrence of lymphoma in rheumatoid arthritis after low dose treatment with MTX (31). The damage produced in culture by MTX is proportional to the growth rate of cells at the time of exposure to the drug (ref.…”

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confidence: 99%

Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate

Chow¹,

Rubin²

1997

A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20-50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9-12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD͞D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD͞D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of conf luence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.