Differential amplification, assembly, and relocation of multiple DNA sequences in human neuroblastomas and neuroblastoma cell lines.

Abstract

DNA amplification, manifested by homogeneously staining regions in chromosomes and by extrachromosomal, double minute bodies, is characteristic of many neuroblastoma cell lines. Sequences recruited from a specific domain on the short arm of chromosome 2 (2p) are amplified in advanced-stage primary neuroblastomas, whereas sequences from distinctly different regions of 2p are amplified in the neuroblastoma cell line IMR-32. Five different DNA segments, which include the oncogene N-myc, three other fragments derived from the homogeneously staining region of the neuroblastoma cell line IMR-32, and a fifth fragment, derived from the neuroblastoma cell line NB-9, showed differential and variable amplification in 24 advanced-stage neuroblastoma tumors out of 112 tested specimens. AU five fragments were mapped within the chromosomal region 2p23-2p25 by three different approaches. However, eight other fragments cloned from the homogeneously staining region of IMR-32 cells, which were not amplified in the tumor tissues examined, were mapped to two more proximal domains of 2p, thousands of kilobases apart from each other and from the chromosomal domain that is amplified in the tumors. These results establish the amplification, to different degrees, of a variable-sized segment of one domain near the terminus of 2p in advanced neuroblastomas. These tumors might ultimately be distinguished according to the pattern of amplification of DNA segments within this domain. The data presented also indicate the existence of a new and complex amplification mechanism in at least one neuroblastoma cell line (IMR-32), which involves not only relocation of DNA from specific genomic domains but also the formation of novel units by splicing together very distant DNA segments.

Characterization of DNA rearrangements of N‐myc gene amplification in three neuroblastoma cell lines by pulsed‐field gel electrophoresis

“…Our studies with PFG analysis provided information on the long-range organization of amplified units: DNA rearrangements had occurred relatively close to the Nmyc gene, however amplification units were much longer and homogeneous. Based on our observations and the data of Shiloh et al [23], the connection of several DNA fragments might be necessary in ~ some cases for amplification of DNA fragments. Such rearrangements should have occurred before, not during amplification.…”

“…Shiloh et al [23] demonstrated that at least three DNA fragments derived from different chromosome loci were simultaneously amplified in HSR of IMR-32. Since they employed in situ hybridization, it was unclear whether these DNA fragments were connected to each other in the same amplification unit.…”

Co-amplification of a novel gene, NAG, with the N-myc gene in neuroblastoma

“…Thus, N-myc is considered the main factor in driving the selective pressure for ampli®cation. However, a number of investigators have found that rearrangements are common in the neuroblastoma amplicon (Akiyama and Nishi, 1991;Amler and Schwab, 1992;Shiloh et al, 1986), suggesting that there may be additional selective pressure for the inclusion or exclusion of other genes. It is also noteworthy that some studies have shown that not all tumors with N-myc ampli®cation are associated with a bad prognosis; for example, 50% of stage II tumors with N-myc ampli®cation have a good outcome De Bernardi et al, 1995).…”

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

“…Previous work has shown that there are usually large differences between the amplified units in independently selected highly amplified mutants (2,5,12,34,36). On the contrary, the highly amplified units within each line are relatively homogeneous.…”

“…In most cases, the results point to remarkable differences between the sequences coamplified in independent highly amplified mutants: the amplified units include a region comprising the selected gene and sequences immediately surrounding it in the wild-type line, but also include sequences which are amplified uniquely in each cell line (2,5,12,34,36,43). Thus, during the amplification process, pieces of DNA from different parts of the genome are joined by recombination to form novel structures and novel joints (new restriction fragments).…”