[37 Transgenic Shuttle Vector Assays for Determining Genetic Differences in Oxidative B Cell Mutagenesis in Vivo

By Klaus Felix, Lynne D. Rockwood, and Siegfried Janz

Because tumorigenesis is an in vivo phenomenon that can be evaluated optimally only in in vivo studies, it has been postulated that attempts to link tumor development with mutagenesis may be most informative when mutagenesis can also be assessed in vivo. We have chosen two transgenic shuttle vector assays, A LIZ (Big Blue) and pUR288 (placZ), to explore in mice the role of oxidative B cell mutagenesis in the development of the malignant plasma cell tumor, plasmacytoma (PCT). Here we provide a brief introduction to the putative role of oxidative stress during mouse plasmacytomagenesis (PCTG) and describe the utility of the XLIZ and pUR288 assays for determining oxidative B cell mutagenesis in vivo. In addition, we introduce a genetic system that employs congenic mouse strains to associate the susceptibility to PCT development with the genetic control of oxidative mutagenesis in the B cell compartment.

BALB/c Plasmacytomas

Our interest in oxidative mutagenesis in B lymphocytes stems from our efforts to learn about the pathogenesis of inflammation-induced PCTs in mice. PCTs are immunoglobulin-producing neoplasms of terminally differentiated

B lymphocytes, that is, plasma cells. The tumors can be induced in genetically susceptible, inbred BALB/c (B/c) mice by intraperitoneal injections of a variety of proinflammatory agents, including the C19 isoalkane pristane (2,6,10,14-tetramethylpentadecane). Treatment with pristane provokes the formation of the pristane granuloma, a chronic inflammatory tissue in the peritoneal cavity. The granuloma is mainly composed of macrophages and further characterized by extensive infiltrates of neutrophils. The ability of macrophages and neutrophils to secrete copious amounts of reactive oxygen intermediates (ROIs) has long raised suspicions that a pathogenetic link exists between oxidative mutagenesis and PCT development. However, the exact nature of this link has been difficult to elucidate because PCTG is a multifactorial process (reviewed in Potter and Wiener1) that has many potential connections to oxidative stress. The following features of PCTG are relevant for contemplating links to inflammation and oxidative mutagenesis:

(1) PCTG takes place in the pristane granuloma. Consequently, PCTs do not develop in normal B/c mice not treated with pristane and, thus, devoid of granuloma;

(2) PCTG is dependent on the chronic inflammatory process in the granulomatous tissue. Treatment of mice with the antiinflammatory agent, indomethacin, abrogates PCT formation in pristane-primed mice without grossly changing granuloma histology. Furthermore, three injections of pristane spaced 2 months apart are considerably more effective in PCT induction than single injections, because three waves of inflammation are generated over a prolonged period of time instead of one wave; (3) PCTG is a multigenic trait that is under tight genetic control. Among a large panel of common inbred strains of mice injected with pristane, B/c was found to be uniquely susceptible to tumor induction, whereas DBA/2N (D2) and many other strains were found to be solidly resistant. PCT-susceptible B/c mice and PCT-resistant D2 mice have since been used as benchmark strains to identify genes that confer susceptibility and resistance to PCT development (R/S genes). R/S genes have been mapped to several regions of the genome, including three loci to the distal part of chromosome 42; (4) PCT susceptibility is incompletely penetrant. At best, only 40-60% of conventionally maintained pristane-treated B/c mice develop PCTs. Certain changes in the environment, for example, in the diet or antigenic exposure of mice, can drastically reduce the tumor incidence; (5) PCTG is a prolonged process that takes on average 220 days to complete; and (6) the histogenesis of PCT development begins with the formation of small clusters of B lymphocytes, lymphoplasmacytoid cells, and plasma cells approximately 30 days after the first injection of pristane. It proceeds with the appearance of plasmacytic foci (compact aggregates of at least 50 plasma cells in one tissue site ~75 days after pristane administration), and, in some cases, megafoci (aggregates of several hundred to a thousand atypical plasma cells ~150 days after pristane administration)

1 M. Potter and F. Wiener, Carcinogenesis 13, 1681 (1992).

2 S. Zhang, E. S. Ramsay, and B. A. Mock, Proc. Natl. Acad. Sci. U.S.A. 95, 2429 (1998).

before culminating in incipient PCTs at 150 to 300 days after pristane administration. In the course of this prolonged oncogenic process, B cells are thought to undergo malignant transformation in close proximity to, or possibly by direct cell-to-cell contact with, macrophages and neutrophils, two significant sources of ROIs in situ. We hypothesized that B cells may become targets of oxidative DNA damage and mutagenesis if the exposure to ROIs overwhelmed their antioxidative defense and DNA repair capacity. If so, the pristane granuloma may function as a catalyst of PCTG by providing an environment of oxidative mutagenesis. We further postulated that D2-typical R genes and B/c-typical S genes may contribute to the phenotypes of PCT resistance and susceptibility by minimizing and increasing, respectively, the levels of oxidative mutagenesis in the B cell compartment.

A.LIZ and placZ Congenies

The /.LIZ (Big Blue) and pUR288 (placZ) mutagenesis assays are based on shuttle vectors (kLIZ and pUR288, respectively) that have been inserted as inheritable transgenes into the germ line of C57BL/6 (B6) mice. A.LIZ has been integrated as a 40-copy concatemer in the central portion of chromosome 4 near Tyrpl (38 cM). The B6-A.LIZ mouse was developed by J. Short and colleagues at Stratagene (La Jolla, CA).3 pUR288 (line 60) is somewhat unusual with regard to transgene integration, as two 10-copy concatemers have been incorporated on chromosomes 3 and 4. The pUR288 mouse was developed by J. Vijg and associates.4'5 We transferred the shuttle vectors from their original B6 background onto the two benchmark strains for genetic studies of PCTG: PCT-susceptible B/c mice and PCT-resistant D2 mice. Strain C.D2-Idhl-Pep3, a B/c mouse congenie for a 41,2-cM-long D2-derived portion of chromosome 1 spanning Idhl and Pep3, was chosen as an additional recipient because it demonstrated the unusual phe-notype of hypersusceptibility to PCT development (~75% tumors 300 days after the first injection of pristane).6 To generate the envisioned congenies, a facilitated backcross protocol was employed that combined detection of the A.LIZ and pUR288 transgenes with selection for recipient-type paternal chromosomes by means of simple sequence length polymorphic markers detectable by polymerase chain reaction (PCR). Of importance, transgene-positive offspring considered for further breeding were routinely tested for rescue efficiency of the shuttle vector. This ensured that only animals in which the mutagenesis assay worked properly were chosen for the next-generation backcross. We highly recommend this control

3 S. W. Kohler, G. S. Provost, A. Fieck, P. L. Kretz, W. O. Bullock, D. L. Putman, J. A. Sorge, and J. M. Short, Environ. Mol. Mutagen. 18, 316 (1991).

4 J. A. Gossen, W. J. de Leeuw, A. C. Molijn, and J. Vijg, Biotechniques 14, 624 (1993).

5 M. E. Boerrigter, M. E. Dolle, H. J. Martus, J. A. Gossen, and J. Vijg, Nature (London) ill, 657 (1995).

6 M. Potter, E. B. Mushinski, J. S. Wax, J. Hartley, and B. A. Mock, Cancer Res. 54, 969 (1994).

C57BL/6-XLIZ "Big Blue"

C57BU6-pUR288 "placZ"

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