David Siwarski

Genomic instability in MycER-activated Rat1A-MycER cells

The deregulated expression of c-Myc protein is associated with the non-random locus-specific amplification of the dihydrofolate reductase (DHFR) gene. This study was performed to determine whether additional chromosomal aberrations occur when c-Myc protein levels are up-regulated for prolonged periods. To this end, we have used Rat1A-MycER cells, which allow the experimental regulation of Myc protein levels. We examined the genomic stability of Rat1A-MycER cells cultivated in either the absence or the presence of estrogen, which reportedly activates the chimeric MycER protein in these cells. Following prolonged periods of MycER activation, Rat1A-Mycer cells exhibited irreversible chromosomal aberrations. The aberrations included numerical changes, chromosome breakage, the formation of circular chromosomal structures, chromosome fusions, and extrachromosomal elements.

Double producers of kappa and lambda define a subset of B cells in mouse plasmacytomas

Rearrangement of the light chain locus is believed to be an ordered process in which Iglambda rearrangements only occur if Igkappa rearrangements are found to be non-productive or self-reactive. Secondary rearrangements of the B-cell receptor (BCR) have shown, however, that rescue of abortive Igkappa rearrangements or autoreactive B cells can be achieved through receptor editing using upstream V-regions as the template sequences. Since secondary rearrangement can occur in the periphery, possibly in a subset of B cells maintaining constitutive Rag activity, it is conceivable that two light chains (kappa:kappa or kappa:lambda) could be expressed in these cells, apparently in violation of allelic exclusion. Previously, we have reported that silicone-induced plasmacytomas (SIPCs) exhibit dual expression and ongoing rearrangements of Igkappa and Iglambda. In this paper, we show by ELISA that both Igkappa and Iglambda are found at the protein level, but are secreted in different amounts. Furthermore, we demonstrate by micro-manipulation and RT-PCR amplification that Igkappa and Iglambda are simultaneously expressed in a single SIPC cell. We propose that these dual-expressing cells, found intermittently in cases of plasmacytomas (PCs), may have originally been immature B cells when transformed but now are maintained as a long-lived mature B cell found infrequently in the tumor population.

Occurrence of a unique MHC class I gene in distantly related members of the genus Mus

There is unequivocal evidence that a relatively nonpolymorphic class I gene (designated Q10) from the Qa region of inbred mice encodes a secreted class 1 molecule. We have used a cDNA probe specific for this gene and an antiserum specific for its secreted protein product to investigate the occurrence and expression of this gene in different species of wild mice broadly representing the entire genus Mus. Evidence is presented that a Q10-like gene has been conserved and is transcribed and translated throughout the genus, suggesting that it serves an important function. However, the data also show that some differences have appeared in this gene over the period of evolutionary time covered by this sampling of wild mice. These results indicate that a specific class I DNA probe isolated from inbred mice can be used to discriminate a particular gene among the multiple class I genes present in other species.

cDNA isolation, expression, and chromosomal localization of the mouse Pcph proto-oncogene

We report here the isolation and characterization of a cDNA from mouse thymus encoding the murine homolog of the protein product of the Syrian hamster Pcph proto‐oncogene. The single open reading frame identified in the cDNA sequence encoded a protein predicted to have 428 amino acids, which shared 93.7% amino acid identity with the Syrian hamster Pcph within the first 412 residues but had a shorter, highly dissimilar C‐terminus. Northern and western analyses revealed that Pcph mRNA and protein were widely distributed in mouse embryo and adult tissues, with the highest expression in adults detected in kidney and liver. The mouse Pcph proto‐oncogene was mapped by linkage analysis to within 3.3±2.3 cM of Pkch‐rs1 on chromosome 12. These data should prove valuable in designing studies to define the cellular function of the Pcph proto‐oncogene. Mol. Carcinog. 26:130–136, 1999.

Structure and Expression of the c-Myc/Pvt 1 Megagene Locus

A chromosomal translocation (Tx) that interrupts the transcription of either c-Myc or Pvt 1 is the principal lesion in many B cell malignancies including Burkitts Lymphoma (BL), AIDs-NHL, mouse plasmacytoma (Pct) and possibly multiple myeloma (MM). There is a restriction associated with this Tx such that only the immunoglobulin (Ig) heavy chain gene is found juxtaposed to c-Myc and only the Ig light chain gene is found juxtaposed to Pvt 1. Over the past several years, our laboratory has been instrumental in the elucidation of the structure of the mouse Pvt 1 locus as a means of understanding the relationship between these two divergent Txs which, nevertheless, produce indistinguishable disease phenotypes. In the mouse, we have identified a uniform Pvt1/Ig Ck fusion product which is consistently found in all tumors harboring Pvt 1 associated Txs. We have recently constructed transgenic mice harboring a translocated Pvt 1/Ck segment in order to determine whether 1). these mice produce the Pvt 1/Ck fusion product 2). these mice are immunocompromised and 3). these mice develop tumors of a B cell origin.

Downregulation of Waf1, C2, C3, and major histocompatibility complex class I loci within an 18-cM region of chromosome 17 in adenovirus-transformed mouse cells.

In this study, the expression of the p53 tumor suppressor gene and the p53‐regulated Mdm2 and Waf1 genes was evaluated in adenovirus (Ad)‐transformed mouse cells. The expected levels of p53 mRNA and protein and Mdm2 mRNA were detected in all transformed cells. However, the level of Waf1 mRNA was markedly reduced in Ad12‐transformed cells and in some Ad5‐transformed cells. Waf1 expression was not reduced in untransformed mouse cells infected with Ad12 or Ad5. Expression of the class 1 major histocompatibility complex (MHC) locus was downregulated in 13 Ad‐transformed cell lines (derived from four different strains of mice) that exhibited reduced expression of Waf1. Waf1 is located in mouse chromosome 17 proximal to the MHC class I locus. To determine whether other chromosome 17 genes were downregulated, the cells were examined for expression of other genetic loci. Of those tested, only the C2 and C3 complement loci were expressed in mouse fibroblasts. Expression of C2 (which is within the MHC) and expression of C3 (which is 15 cM distal to the MHC) were downregulated in those transformed cells in which Waf1 and MHC class I were downregulated. The Ad12‐ and Ad5‐transformed cells that expressed low levels of Waf1, MHC class I, C2, and C3 formed tumors in syngeneic adult mice. These data suggest that the downregulation of multiple genes within the 32 Mb of mouse chromosome 17 that includes the Waf1 locus to the C3 locus occurs in Ad mouse‐cell transformation and may contribute to the tumorigenicity of transformed cells. Mol. Carcinog. 18:213–220, 1997.

A murine antibody to Shigella dysenteriae type 1 employs V-genes that contain a rearranged codon for the λ light chain

The cDNA coding for a hybridoma anti Shigella dysenteriae type 1 antibody (3707 E9) has been cloned, and sequenced. Binding patterns with fragments of the bacterial polysaccharide antigen had already been studied in detail. The VH sequence utilizes the VH441 gene, first identified amongst beta-(1,6)galactan-binding antibodies, while the VL is closely related to the V lambda 1 gene. We found that the VL3707 E9 gene employed a VL-J combinatorial joining leading to a rare Trp-->Leu substitution at position L96.

Double light chain producing lymphocytes: an enigma of allelic exclusion.

The infrequent double light chain producing lymphocyte (DLCPL) is discussed in the context of allelic exclusion. Principally allelic selection rather than allelic exclusion would suggest a role for the DLCPL in the normal B cell population rather than as an aberrance of B cell malignancy. Found primarily in the periphery, it is uncertain at what stage of B cell ontogeny the DLCPL might reside. Nevertheless, through the possible presentation of two functional surface receptors, the DLCPL could be capable of recognizing both self and nonself epitopes.

Expression of c-myc and Pvt-1.

Deregulated expression of c-myc secondary to chromosomal alterations is a constant feature of mouse plasmacytomas, rat immunocytomas and human Burkitt lymphomas (Mushinski, 1988). One feature of deregulated expression of c-myc is that all plasmacytomas have high levels of c-myc transcripts, no matter whether their chromosome 15 was interrupted by a rcpt(12;15) translocation, by a rcpt(6;15) translocation, or by interstitial deletion within chromosome 15.

A novel H-2s class I molecule expressed on a B-Cell leukemia from SJL/J mice

Anti-H-2.33 [(B10.D2 X A)F1 anti-B10.A(5R)], which predominantly contains antibodies recognizing H-2Kb and IAb molecules, was found to be cytotoxic against DMLM 1678, a B-cell leukemia of SJL/J (H-2s) origin. The antiserum precipitated a typical class I (H-2-like) molecule from labeled tumor cell preparations as judged by molecular mass, papain susceptibility and association with beta 2-microglobulin. Sequential immunoprecipitation studies revealed that it was distinct from either H-2Ks or H-2Ds, the 2 molecules expressing the private antigens of the H-2s haplotype. Absorption analysis using congenic mice mapped the gene controlling the expression of the novel molecule telomeric to the S-region within the major histocompatibility complex.