Double minute chromosomes (DMs) are generated as a consequence of DNA amplification, which is a large increase in the numbers of a specific gene and its adjacent DNA on either side, because of their selective replication. Some types of amplification result from specific developmental requirements, such as the synthesis of multiple copies of the large ribosomal RNA genes in Xenopus laevis oocytes (1). This amplification of ribosomal RNA genes meets the enormous needs for ribosomal synthesis during Xenopus oogenesis. More typically, DNA amplification of the type that leads to the appearance of DMs occurs as part of an environmental stress response in cells in culture.
Tissue culture cells exposed to a toxic substance, such as those used in cancer chemotherapy, often eventually become resistant to the substance. One way of achieving a resistant state is to amplify a gene whose product metabolizes the toxic substance into a safer nontoxic form. A typical example is the amplification of the gene encoding dihydrofolate reductase (DHFR), an enzyme involved in thymidine synthesis, in response to methotrexate, which is an inhibitor of DHFR synthesis (see Aminopterin, Methotrexate, Trimethoprim, and Folic Acid). Amplification of the DHFR gene can also be stimulated in response to other environmental conditions, such as UV light and hydroxyurea (2).
Two visible manifestations of the amplification process occur at the chromosomal level. One is the appearance of extended regions in the chromosome known as homogeneously staining regions (HSRs), whose staining characteristics differ from those of normal chromosomal. These HSRs are the sites of DNA amplification (3). The second visible change in chromosomal material is the appearance of DMs that contain the same amplified DNA, but have become extrachromosomal. The relationship between DMs and HSRs is not completely clear, but it is likely that DMs are unstable precursors of HSRs. Human primary tumors contain DMs 90% of the time, 7% contain HSRs, and 3% contain both DMs and HSRs. When cell lines are grown for extended periods of time under drug-resistant conditions, greater numbers of HSRs appear and fewer DMs are found.
DMs are basically acentric extrachromosomal elements that are small fragments of chromosomes. Each DM contains between 1 to 2 megabase pairs of DNA and can replicate itself. Therefore DMs function as minichromosomes . The physical organization of the DNA is that of a supercoiled circle (4). Because DMs are acentric they are progressively lost during cell division, unless they replicate multiple times in any cell cycle. In general they segregate randomly between daughter cells at mitosis, leading to unequal numbers in daughter cells. As many as 20 DMs containing DHFR DNA are found in some methotrexate-resistant cell lines. Oncogenes might also be present in the amplified DNA of DMs and HSRs. For example, the c-Ki-ras oncogene is amplified more than 30fold in DMs and HSRs in certain adrenocortical tumor cell lines (5). Double minute chromosomes are important indicators of abnormal cellular behavior, and they may well reflect an advanced stage of malignancy.
