ABPC 48 cross-reactive idiotopes in BALB/c mice. Natural and levan-induced expression.

Using monoclonal antiidiotypic antibodies, we developed a sensitive binding assay that detects molecules with one or with two idiotopes of the ABPC48 idiotype. ABPC48 cross-reactive idiotypes were thus shown to be present in substantial amounts in sera of nonimmunized mice. Levan binding sites are found on these idiotypes. During the life time of the mice, the natural anti-levan titer increases while ABPC48 idiotypic expression remains constant, suggesting different controls for these two activities. On the other hand, ABPC48 cross-reactive idiotypes participate--as minor components--in the response that follows a deliberate immunization with bacterial levan. This induction process is likely to reflect the selection of idiotopes expressed by the B cell clones preactivated in sera of nonimmunized mice rather than the activation of silent clones. We suggest that a similar situation might explain the reported emergence of ABPC48 idiotypes in animals primed with antiidiotypic antibodies and subsequently stimulated with levan.

The origin of chromosome rearrangements at early stages of AMPD2 gene amplification in Chinese hamster cells.

BACKGROUND: Gene amplification and chromosomal rearrangements are frequent properties of cancer cells, provoking considerable interest in the mechanism of gene amplification and its consequences - particularly its relationship to chromosomal rearrangements. We recently studied the amplification of the gene for adenylate deaminase 2 (AMPD2) in Chinese hamster cells. Using fluorescent in situ hybridization (FISH), we found that early amplification of the AMPD2 gene is based on unequal gene segregation at mitosis, rather than local over-replication. We observed large inverted repeats of the amplified sequences, consistent with an amplification mechanism involving cycles of chromatid breakage, followed by fusion after replication and, in mitosis, the formation of bridges between the fused sister chromatids that leads to further breaks - a process we refer to as chromatid breakage-fusion-bridge (BFB) cycles. Our previous work left open the question of how this mechanism of gene amplification is related, if at all, to the chromosomal rearrangements that generate the dicentric, ring and double-minute (DM) chromosomes observed in some AMPD2-amplified metaphase cells, which are not predicted intermediates of chromatid BFB cycles, although they could be generated by related chromosome BFB cycles. RESULTS: We have addressed this question using FISH with probes for the AMPD2 gene and other markers on the same chromosome. Our results are not consistent with the chromosome BFB cycle mechanism, in which two chromatids break simultaneously and fuse to generate, after replication, a dicentric chromosome. Rather, they suggest that dicentric chromosomes are generated by secondary events that occur during chromatid BFB cycles. Our results also suggest that DM chromosomes are generated by the 'looping-out' of a chromosomal region, generating a circular DNA molecule lacking a centromere; in this case, gene amplification would result from the unequal segregation of DM chromosomes at mitosis. CONCLUSION: We conclude that, at early stages of AMPD2 gene amplification, chromatid BFB cycles are a major source of both 'intrachromosomal' gene amplification and genomic rearrangement, which are first limited to a single chromosome but which can then potentially spread to any additional chromosome. It also seems that, occasionally, a DNA sequence including the AMPD2 gene can be excised, generating a DM chromosome and thus initiating an independent process of 'extrachromosomal' amplification.

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase.

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.

Segregation and rearrangement of coamplified genes in different lineages of mutant cells that overproduce adenylate deaminase.

Four genes encoding proteins designated as W, X, Y1, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Y1 gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes--in a particular order--in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.

Immune response induced by a single or several syngeneic monoclonal antiABPC48 antiidiotypic antibodies: no predominant coexpression of ABPC48 idiotopes.

BALB/c mice were immunized with monoclonal BALB/c antibodies IDA10, IDA16 and IDA17 raised against the BALB/c ABPC48 myeloma protein. Several procedures of immunization--copolymers with lipopolysaccharide or keyhole limpet hemocyanin, simultaneous or sequential injections of different IDAs--were performed in an attempt to orient the immune response towards the production of ABPC48-like idiotypes. We used a binding assay which identifies two idiotopes on the same molecule to measure the population of antibodies induced in these responses. The expression of ABPC48 cross-reactive idiotypes in immune sera was analyzed. The results show that, with all immunization protocols, immune responses to different monoclonal antiidiotypic antibodies are mostly independent of each other: the coexpression of ABPC48 idiotopes, either private or recurrent on the induced antibodies, is rarely found; it makes it difficult to discriminate by a serological approach between cross-reactive idiotypes and anti-antiidiotypic antibodies. We discuss the interest of combining molecular and serological approaches to identify these two populations of antibodies.

A single VH-gene associated with a variety of D- and J-segments encodes for a large family of ABPC48-related antibodies induced by antiidiotypic immunization.

Two series of monoclonal antibodies have been obtained from BALB/c mice immunized against two antiABPC48 antiidiotypic antibodies. They are divided into two serologically different classes. Class I antibodies bind only the immunizing antibody; class II antibodies display a broad binding capacity to various antiidiotypic antibodies, and some bind levan, as does ABPC48. Northern blot analyses and partial mRNA sequencing show that all class II antibodies express the VH-gene coding for ABPC48 and UPC10 antilevan antibodies associated with a variety of D- and J-segments. The third hypervariable region of the sequenced antibody with antilevan activity is structurally related to that of ABPC48 and UPC10 antibodies but has a different genetic origin. This study indicates that the identification of idiotype-related antibodies arising from antiidiotypic immunization may be misleading, if based on their antigen-binding properties; and it stresses the importance of structural approaches for the analysis of regulatory mechanisms ruling immune responses.

Specificity and idiotypic analysis of monoclonal antibodies directed against the MOPC460 idiotype.

Eight syngeneic anti-idiotypic hybridomas (IDMs) have been obtained against the BALB/c myeloma protein MOPC460 which displays anti-TNP activity. The study of their anti-idiotype specificity allowed us to distinguish them into two groups which define the presence of at least two idiotypic determinants or idiotopes in the MOPC460 idiotype. The biochemical analysis of the monoclonal antibodies is consistent with this dichotomy. This analysis, in fact, showed a striking correlation between anti-idiotypic specificity and biochemical characteristics of the monoclonal antibodies. Consequently, the idiotypic specificities of three of these hybridomas were studied. In accordance with what is expected, our results clearly indicate a strong idiotypic similarity for hybridomas belonging to the same group and a lack of idiotypic cross-reactivity.

A new rapid rosette-forming cell micromethod for the detection of antibody-synthesizing hybridomas.

The method reported here describes a microrosetting assay which allows the early detection of antibody-synthesizing hybridomas. This method is specific, requires very few hybridoma cells and avoids artifacts resulting from the presence of antibodies secreted by spleen cells in the primary hybridoma cultures. It is easy to perform and allows the screening of more than one hundred independent clones within a few hours.

A glutathione depletion selectively imposed on mu glutathione S-transferase overproducing cells increases nitrogen mustard toxicity.

Glutathione (GSH) contributes to the detoxification of anticancer drugs through the operation of specific glutathione S-transferases (GST) and innate, or acquired, overexpression of this enzyme family has been frequently observed in tumor cell lines. In the GMA32 line of Chinese hamster fibroblasts, we showed that GSH starvation produced by exposing cells to buthionine sulfoximine (BSO) increased the toxicity of chlorambucil and melphalan, but not that of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), cisplatine and doxorubicin. This indicates that efficient mechanisms of detoxification using GSH operate for chlorambucil and melphalan, but not for the other drugs in these cells. We then showed that GSH depletion could be selectively and transiently induced in the mu GST overexpressing cell line derived from GMA32, HC474, by exposing cells to substrates specific to the overexpressed isozyme. Exposing cells to such a substrate, trans-stilbene oxide, does not alter the sensibility of GMA32 cells to melphalan and chlorambucil, but increases that of HC474 cells to these drugs, to an extent comparable to that obtained with BSO. This observation highlights the possibility of exploiting GST overexpression, a frequent feature of tumor cells, to selectively sensitize these undesirable cells to anticancer drugs.

The splenic pool of mouse stem cells: in vitro differentiation and expression of the BP-1 alloantigen on cells of the lymphoid and myeloid lineages.

The relative paucity of data about the development of the stem cell pool present in the spleen prompted this study. During in vitro cultures of B-enriched lymphocytes from mouse spleens and in the presence of a culture supernatant of WEHI-3 cells (WEHI-SUP), a population of cells expressing the BP-1 antigen appears progressively, reaches an optimal size 8 days after initiation of the culture, and disappears on day 28. In 8-day-old cultures, a minor population of cells bearing both BP-1 and B220 can be detected. The growth of this cell population, with characteristics of the B lymphoid lineage (pro-B), is strictly dependent on the presence of WEHI-SUP in the medium. After 2 weeks of culture, the BP-1 antigen is expressed on a cell population, which is essentially constituted of B220-, polynuclear cells. The BP-1 antigen, which is considered as characteristic of early cells of the B lymphoid lineage, can therefore also be expressed on cells of the myeloid lineage. The injection of BP-1+ or B220+ cells in irradiated mice can hardly reconstitute their B cell pool, whereas BP-1- and B220- cells are much more efficient in vivo progenitors of this cell lineage.

Gene amplification mechanisms: the role of fragile sites.

We studied the early stages of gene amplification in a Chinese hamster cell line and identified two distinct amplification mechanisms, both relying on an unequal segregation of gene copies at mitosis. In some cases, a sequence containing the selected gene is looped out, generating an acentric circular molecule, and amplification proceeds through unequal segregation of such extrachromosomal elements in successive cell cycles. In other cases, the accumulation of intrachromosomally amplified copies is driven by cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere, which leads to bridge formation and further break in mitosis (BFB cycles). We showed that some clastogenic drugs specifically trigger the intrachromosomal amplification pathway and strictly correlated this induction of BFB cycles to the ability of these drugs to activate fragile sites. In three model systems, we also established, that the location of centromeric and telomeric fragile sites relative to the selected genes determines the size and sequence content of the early amplicons.

Reversion in Chinese hamster lines amplified at the AMPD2 locus: spontaneous and benzamide-stimulated gradual loss of amplified alleles of marker genes.

The HC47 and HC474 cell lines of Chinese hamster fibroblasts resist coformycin through the intrachromosomal amplification of the AMP deaminase 2 (AMPD2) gene. Due to the coamplification of a mu glutathione S-transferase (GST) gene, these mutant lines are more sensitive than GMA32 wild-type parental cells to buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis. This property was exploited to select revertants of amplification from HC474 cells. Reversion in that line is frequently a gradual process that does not involve extrachromosomal intermediates. The terminal products of this process are commonly cells with a complete deletion of the amplified allele of marker genes and are therefore haploid for these loci on the homologous chromosome. Exposing HC474 cells to benzamide (BA), an inhibitor of polyADP-ribosylation, increased the recovery of revertants to an extent allowing the detection of reverting cells without BSO selection. This effect of BA was used to isolate revertant cells from the HC47 line that is extremely stable and to demonstrate that the mechanism of gradual reversion also occurs in this line. The gradual deletion of amplified copies within the chromosomes suggests that breakage-fusion-bridge (BFB) cycles drive this process.

The evolution of the amplified adenylate deaminase 2 domains in Chinese hamster cells suggests the sequential operation of different mechanisms of DNA amplification.

Fluorescent in situ hybridization was used to localize the adenylate deaminase 2 (AMPD2) genes and flanking sequences on the chromosomes of the Chinese hamster line GMA32 and to study the distribution of additional copies of these genetic sequences in amplified mutants selected at several early stages of the amplification process. The synteny of AMPD2 genes and MDR1 genes, located on chromosomes 1, was demonstrated; in GMA32 the existence of a rearrangement positioning the two AMPD2 genes at different distances from the telomeres was disclosed. Using this structural marker, we showed that the amplified copies distribute along only one of the chromosomes 1. Their organization in different cells of clonal mutant populations at a very early stage of amplification was extremely heterogeneous; classes of organization could be recognized however. Their quantitative distribution at this stage and in cells which went through 10 more division cycles suggests an evolution pathway common to the mutant clones under study: as a rule, tandems of few units of identical and very large size (47 Mb) appear to be the first detected product of amplification; this organization is progressively overtaken by structures with more units of reduced and irregular size, while, in a growing number of cells, clusters of much shorter units can be observed. The nature of segregative amplification mechanisms operating in these processes and the possible involvement of replicative ones are discussed.

[Gene amplification and chromosomal rearrangements during acquisition of cellular resistance to the antimetabolite coformycin]. / Amplification génique et réarrangements chromosomiques lors de l'acquisition de la résistance cellulaire à un antimétabolite, la coformycine.

We studied the early stages of gene amplification in a Chinese hamster cell line and we show that two distinct mechanisms can operate at a single locus. Both of them rely on an unequal segregation of gene copies at mitosis. We conclude that cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere that lead to further breaks in mitosis, have a key role in the coupling of gene amplification and genome remodeling. Rearrangements are first limited to a single chromosome but can then potentially spread to any additional chromosome. Occasionally, a sequence containing the selected gene can be looped out, generating a "double minute" and thus initiating an independent process of extrachromosomal amplification.