Drug-selected intrachromosomal gene amplification by breakage-fusion-bridge (BFB) cycles is well documented in mammalian cells, but factors governing this mechanism are not clear. Here, we show that only some clastogenic drugs induce drug resistance through intrachromosomal amplification. We strictly correlate triggering of BFB cycles to induction of fragile site expression. We demonstrate a dual role for fragile sites in intrachromosomal amplification: a site telomeric to the selected gene is involved in initiation, while a centromeric site defines the size and organization of early amplified units. The positions of fragile sites relative to boundaries of amplicons found in human cancers support the hypothesis that fragile sites play a key role in the amplification of at least some oncogenes during tumor progression.

Two‐colour in situ hybridization with probes for two co‐amplified markers located several megabases apart on chromosome 1 has been used to analyse early stages of adenylate deaminase 2 (AMPD2) gene amplification in Chinese hamster cells. In the amplified chromosomal structures, the distribution of hybridization spots identifies megabase‐long inverted repeats. Their organization is remarkably well accounted for if breakage‐fusion‐bridge cycles involving sister chromatids drive the amplification process at these early stages. During interphase the markers often segregate into distinct nuclear domains. Many nuclei have bulges or release micronuclei, carrying several copies of one or both markers. These observations indicate that the amplified units destabilize the nuclear organization and eventually lead to DNA breakage during interphase. We propose a model in which interphase breakage has a role in the progression of gene amplification.

Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB ؋ NZW)F 1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab)2 and Fab fragments, covalently coupled to f luorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or antiperoxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes.A long time ago, it was reported that human IgG from systemic lupus erythematosus patients with high titers directed against nuclear ribonucleoproteins and/or DNA were able to penetrate into living cells and to reach the nucleus (1). More recent studies of murine anti-DNA autoantibodies confirmed these observations and disclosed that different penetrating antibodies exhibited diverse behaviors and characteristics (2-7). In this study, we prepared several penetrating IgG anti-DNA mAbs from the spleen of a (NZB ϫ NZW)F 1 lupus mouse and examined their specificities and their abilities to act as vectors of haptens, proteins, polynucleotides, and plasmids. MATERIALS AND METHODSMice and Cell Lines. (NZB ϫ NZW)F 1 hybrids and BALB/c mice were bred in the Institut Pasteur animal facilities. Cells used were from different species and from various tissues as follows: PtK2 (Potoroo kidney fibroblasts) or CCL-39 (hamster lung), 3T3 (mouse embryo fibroblasts), and HEp-2 (human larynx carcinoma). All cells were from the American Type Culture Collection and were cultured in RPMI 1640 medium (or in DMEM for CCL-39) containing 10% heat-inactivated calf serum and supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, and antibiotics (complete culture medium) at 37°C in a humidified atmosphere of 5% CO 2 /95% air.mAbs. Spleen cells from a 9-month-old nonimmunized (NZB ϫ NZW)F 1 mouse were fused with P3.X63Ag8 myeloma cells by the method of Köhler and Milstein (8), and hybridomas were selected in hypoxanthine/azaserine medium. Supernatants were tested by ELISA on double-stranded (ds) DNAcoated plates with ␤-galactosidase-labeled anti-Fc␥ conjugate prepared from sheep antiserum (9). Isotypes were determined by using anti-IgG1-, -IgG2a-, -IgG2b-,...

The amplified DNA of HC50474, a Chinese hamster fibroblast cell line selected in three steps for high resistance to coformycin, consists chiefly of 150 copies of a large inverted duplication including the adenylate deaminase gene. Most if not all of these units are more than 2 x 120 kb long. The inverted duplication was first detected in the cells recovered from the second selection step, at the same chromosomal location as the first step amplified units. Its formation and amplification appear to be coupled since the second step cell line already contained 40 copies of this novel structure. Reamplification of the inverted duplication occurred at the third step of selection concomitant with the loss of amplified DNA acquired during the first step. The head‐to‐head junction has been formed by recombination within a recombinational hotspot described previously [Hyrien, O., Debatisse, M., Buttin, G. and Robert de Saint Vincent, B. (1987) EMBO J., 6, 2401‐2408]. Sequences at the joint and in the corresponding wild‐type region reveal that the crossover sites, one of which occurs in the putative promoter region of B2 repeat, are located at the top of significant stem‐loop structures and that patchy homologies between the parental molecules on one side of the breakpoints allow alignment of these crossover sites. We present a model which explains the formation and amplification of this and other large inverted duplications by errors in DNA replication.