Inhibition of plasmacytoma development in BALB/c mice by indomethacin.

Indomethacin given continuously in the drinking water (20 micrograms/ml) to BALB/cAn pi mice during the latent period of pristane-induced plasmacytoma development dramatically reduced the plasmacytoma incidence from 34.9 to 2.2%. Additionally, indomethacin given from day 0 to 120 or begun as late as 60 d after a single injection of 1.0 ml pristane was also highly effective in reducing the development of plasmacytomas. Indomethacin treatment did not prevent the formation of a peritoneal inflammatory exudate or peritoneal oil granulomatous tissue, although it had a mild inhibitory effect on the intensity of the cellular inflammation, particularly after extensive treatment of greater than 100 d. Indomethacin treatment reduced the incidence of arthritis by 50%. A major effect of indomethacin treatment was a reduction in the appearance of microscopic plasmacytomas that appear in the oil granuloma before plasmacytomas can be detected by routine sampling of the peritoneal exudate. Between days 116 and 181, 16 of 20 mice given 0.5 ml pristane were found to have foci of plasmacytoma cells, while only 2 of 20 indomethacin-treated mice had foci-containing plasmacytoma cells. The number of mice with microscopic foci in the pristane-treated group greatly exceeded the expected incidence of plasmacytomas (22%) at this dose of pristane. The growth of primary plasmacytomas in transplant that is dependent on the pristane-conditioned peritoneal environment was not inhibited by indomethacin treatment. The role of indomethacin in inhibiting plasmacytoma development was not established; two possibilities are that it inhibits production of mutagenic and tissue destructive oxidants by inflammatory cells, and it inhibits prostaglandin synthesis and intracellular production of oxidant biproducts.

Effect of MuLV-related genes on plasmacytomagenesis in BALB/c mice.

The role of spreading somatic cell infections with ecotropic MuLV viruses in the induction of plasmacytomas in BALB/cAN pi mice was determined by constructing congenic mice that lacked the gene locus Cv that codes for ecotropic virus. DBA/2 mice that lack Cv on chromosome (chr) 5 carry a closely linked gene Rmcfr that determines resistance to infection with mink cell focus-forming viruses (MCF). Rmcfr was retrogressively back-crossed onto BALB/c for six successive generations to produce N6 mice. N6 mice were mated to each other to produce BALB/c.DBA/2 Rmvfr/Rmcfr homozygotes. This stock of mice lacked Cv, as demonstrated by DNA hybridization and were as fully susceptible to developing plasmacytomas as the parental BALB/c. A second congenic stock BALB/c.DBA/2 Rmcfr/Rmcfr Fv-1n/Fv-1n was also developed, but the mice of this stock showed a reduced incidence of plasmacytomas, as did BALB/c.DBA/2 Fv-1n/Fv-1n mice. These findings indicated Fv-1 or a gene closely linked to it conferred partial resistance to plasmacytomagenesis. In constructing the BALB/c.DBA/2 Fv-1n/Fv-1n stock, a "control" congenic BALB/c.DBA/2 Fv-1b/Fv-1b was also developed at N6. Surprisingly, this stock carried the Qa2+ trait. These mice were also partially resistant to plasmacytomagenesis, suggesting a gene on chromosome 17 (the location of Qa2) or a gene located elsewhere that regulates Qa2 expression is linked to a gene controlling partial resistance to plasmacytoma development.

High resolution banding analysis of the involvement of strain BALB/c- and AKR-derived chromosomes No. 15 in plasmacytoma-specific translocations.

Plasmacytomas were induced in (BALB/c X AKR 6;15) X BALB/c backcross mice where one of the BALB/c-derived chromosomes No. 15 was replaced by the AKR(6;15)-derived Robertsonian 6;15 chromosome. (BALB/c X AKR 6;15)F2 mice that were homozygous for Rb 6;15 were mated to BALB/c mice. Plasmacytomas were induced in the progeny by intraperitoneal injection of pristane. The cytogenetic marker permitted the distinctive identification of the two chromosome 15 homologues, including the distal segment involved in the plasmacytoma-specific translocations. 7 of the 10 plasmacytomas contained the typical t(12;15) translocation. The BALB/c-derived 15 chromosome served as the donor of the translocated segment in six of them. In the seventh, the Rb 6;15 chromosome of the AKR strain was the donor. The remaining three tumors contained the same type of intrachromosomal rearrangement. It arose by the pericentric inversion of the Rb 6;15 chromosome, leading to a variant plasmacytoma-associated rcpt (6;15) translocation. Unlike the usual 6;15 variant that arises by a reciprocal exchange between two separate chromosomes, it was generated by an exchange of the distal segments of a single chromosomal element. High resolution banding analysis of the tumors showed that all translocated breakpoints on chromosomes 15, 12, and 6 were identical with the previously described breakpoints characteristic for the typical 12;15 and the variant 6;15 translocation in murine plasmacytomas. It is known that the distal segment of chromosome 15 carries the c-myc oncogene (23). The PC-associated translocations cut across the 5'-exon of c-myc in the majority of the cases (24,26). The severed oncogene is transposed to the Ig-region on the recipient chromosome. Since the BALB/c strain is highly sensitive to PC-induction, we were interested to examine the question whether its chromosome 15 is preferred as the oncogene donor in AKR X BALB/c backcross mice that carry cytogenetically distinguishable 15 chromosomes. Our results show that this is not the case, since the same segment of the AKR-derived chromosome 15 could also serve in the same capacity. This is in contrast with T cell leukemogenesis where we have previously found that the trisomization-associated duplication of chromosome 15 occurred in a highly asymmetrical fashion, depending on the donor strain of No. 15 (9-11).

Avian v-myc replaces chromosomal translocation in murine plasmacytomagenesis.

Deregulation of c-myc expression in association with chromosomal translocations occurs in over 95% of murine plasmacytomas, rat immunocytomas, and human Burkitt lymphomas. Infection with a murine retrovirus (J-3) containing an avian v-myc rapidly induced plasmacytomas in pristane-primed BALB/cAn mice. Only 17% of the induced plasmacytomas that were karyotyped showed the characteristic chromosomal translocations involving the c-myc locus. Instead, all of the translocation-negative tumors demonstrated characteristic J-3 virus integration sites that were actively transcribed. Thus, the high levels of v-myc expression have replaced the requirement for chromosomal translocation in plasmacytomagenesis and accelerated the process of transformation.

Peritoneal plasmacytomagenesis in mice: comparison of different pristane dose regimens.

Plasmacytomas were induced in inbred BALB/c pi mice by the ip injection of pristane with 4 different dose schedules. Three 0.5-ml doses (1.5 ml) given at 2-month intervals gave an average yield of 61% plasmacytomas in 6 experimental groups with a range from 51 to 71%; a single 1-ml dose gave an average yield of 42% plasmacytomas in 5 experimental groups with a range from 37 to 45%; and a single 0.5-ml dose gave an average of 22% from 3 experiments involving young mice with a range from 14 to 26%. Two 0.5-ml doses given at various intervals from 14 to 300 days gave yields of plasmacytomas that usually but not always were greater than that obtained with a single 0.5-ml dose of pristane. When the second injection of pristane was delayed as long as 180 days, a strong additive effect over that observed with 0.5 ml alone was obtained. The plasmacytomas developed in mice given the second dose 180 days after the first, with virtually the same latent period as observed with a single 1-ml dose. No plasmacytomas were found in 200 BALB/c pi mice inoculated with corn oil, aluminum hydroxide, or very small doses of pristane (i.e., 0.05 ml). The minimal latent period for plasmacytoma development is about 120 days. The median latent period ranged from 180 to 250 days in the groups of mice that received 3 0.5-ml injections of pristane. In a single experiment pristane freed of UV-absorbing materials was as potent as commercial grade pristane in inducing plasmacytomas.

Identification of two genes on chromosome 4 that determine resistance to plasmacytoma induction in mice.

BALB/cAn mice are highly susceptible to the induction of plasmacytomas (PCTs) by the i.p. injection of paraffin oils, whereas DBA/2 mice are solidly resistant. To search for genes that control the dominant resistant phenotype of DBA/2, BALB/c.DBA/2 (C.D2) congenic strains were constructed, and the susceptibility and resistance to PCT development were determined. PCT formation takes place over an extended period of 365 days but begins morphologically in focal proliferations of atypical plasma cells (foci) in the reactive oil granuloma that forms on mesenteric surfaces. Cells from some of these foci spread to other locations in oil granuloma tissue, forming new foci. Mice that develop six or more foci appear to be progressing towards eventual overgrowth and replacement of all peritoneal tissues with PCT cells. From Days 100 to 250, between 28 and 56% of PCT-susceptible BALB/cAn mice had 6 or more foci, whereas less than 5% of resistant DBA/2, BALB/c x DBA/2 F1 (hereafter called CD2F1), C57BL/6, and BALB/cJ mice had 6 or more foci. Four CD2 congenic strains carrying D2 alleles of genes on chromosomes other than chromosome 4 were highly susceptible. Between 0 and 20% of the mice in C.D2-Chr 4 congenic strains C.D2-MIA, C.D2-TF3, C.D2-Fv-1n/n, C.D2-Pnd7, C.D2-Lgm-1A, C.D2-Lgm-1B, C.D2-Lgm-1C, and C.D2-Lgm-1H developed 6 or more foci from 125 to 260 days, indicating resistance. The segments of DBA/2 chromosome 4 chromatin in C.D2-Fv-1n/n and C.D2-Pnd7 were discontinuous with those in C.D2-TF3, C.D2-Lgm-1A, C.D2-Lgm-1B, C.D2-Lgm-1C, and C.D2-Lgm-1H, indicating there are at least two genes (Pctr1 and Pctr2) in the distal half of this chromosome that confer resistance. Pctr1 is located between Ifa and D4Rck41, and Pctr2 is between Tnfr-1 and Pkcz. Each locus acting alone distinctly conferred a partial resistant phenotype. Pctr1 and Pctr2 did not appear to prevent the formation of clonal foci but did appear to limit the ability of the plasma cells in foci to acquire greater autonomy; thus, these genes affect tumor progression.

v-myc and v-raf act synergistically to induce B-cell tumors in pristane-primed adult BALBC mice.

In a variety of systems, evidence is accumulating which suggests that neoplastic transformation requires the action of two or more genes such as mutated or over-expressed proto-oncogenes. To determine whether the cytoplasmic serine/threonine kinase oncogene raf could complement a deregulated myc gene and induce tumors in adult mice, BALBC mice were primed with an intraperitoneal (ip.) injection of mineral oil (pristane) and then given an ip. injection of a retroviral construct, J1, J2 or J5, which expresses either v-raf (J1), v-myc (J5) or both (J2). The J1 virus induced no tumors in 150 days in 38 mice, except for 5 helper virus-associated T-cell lymphomas. Under identical conditions the J5 virus, which expresses only v-myc, induced exclusively monocytic neoplasms in 93% of 15 mice. The J2 virus expresses both v-myc and v-raf and caused equal numbers of monocytic and B cell tumors in 66% of 30 mice. Under these conditions, it appears that v-raf expression acts synergistically with v-myc to induce the transformation of B cells, which neither oncogene could do alone. The J3 virus, which originally contained a complete v-myc and an inactivated v-raf, can induce tumors of later stage B cells (plasmacytomas, Potter et al., 1987). Recent studies of virus recovered from these plasmacytomas (called the J3V1 virus, Troppmair et al., 1989) show that the J3 virus has undergone deletions which have reactivated v-raf in a mutated form. Only J3V1, not J3, induced tumors in vivo. Our data presented here corroborate Troppmair et al. and extend Potter et al. (1987) which reported that J3 (presumably J3V1) induced 10% myeloid tumors and 90% plasmacytomas. In light of the discovery, our J2 and J3 data indicate that in combination with the same form of v-myc, different forms of v-raf induce different spectra of tumors.

Genetics of susceptibility to pristane-induced plasmacytomas in BALB/cAn: reduced susceptibility in BALB/cJ with a brief description of pristane-induced arthritis.

BALB/cAn and BALB/cJ inbred strains of mice were separated 43 yr ago, but are still very closely related because they carry the same allelomorphs at many loci. When injected 3 times i.p. with 0.5 ml of pristane (2, 6, 10, 14 tetramethylpentadecane) 11% of strain BALB/cJ develop plasmacytomas in contrast to 61% in BALB/cAn sublines. BALB/cJ first injected with pristane as neonates also developed a reduced number of plasmacytomas as compared with BALB/cAn. About 20% of pristane-treated BALB/cAn and 70% of pristane-treated BALB/cJ develop arthritis, which first appeared in the ankle joints after a latent period of 4 mo or more.

An altered v-raf is required in addition to v-myc in J3V1 virus for acceleration of murine plasmacytomagenesis.

We have isolated and molecularly cloned a highly pathogenic virus variant, J3V1, from murine plasmacytomas induced by a combination of pristane and the weakly transforming recombinant retrovirus J3. J3 virus is a derivative of the v-raf/v-myc-carrying J2 virus that was generated by a frameshifting deletion inactivating v-raf in J2. J3V1 contains an additional deletion of 334 base paris in gag, which restores the correct reading frame for v-raf and results in the expression of a p57 gag-raf fusion protein. Reactivation of v-raf in J3 is required for efficient plasmacytoma acceleration in pristane-conditioned BALB/cAn mice.

The plasmacytoma resistance gene, Pctr2, delays the onset of tumorigenesis and resides in the telomeric region of chromosome 4.

Mouse plasmacytomas share pathogenetic features in common with both multiple myeloma and Burkitt's lymphoma in humans. Susceptibility to plasmacytoma induction by intraperitoneal pristane in mice is controlled by multiple genes. At least two of these genes reside on mouse chromosome 4 in regions of the genome sharing linkage homology with human chromosomes 9p21, 1p32, and 1p36. A series of congenic strains recombinant for regions of mouse chromosome 4 in the vicinity of the Pctr2 predisposition locus were created and typed for their tumor susceptibility/resistance phenotypes. These strains were derived by introgressively backcrossing alleles from resistant DBA/2 mice onto the susceptible BALB/cAnPt background. Six resistant and two susceptible strains were allelotyped for 10 genes and 49 random DNA markers to identify the smallest region of overlap in the resistant strains. These studies have determined that the Pctr2 locus resides in either a 500-kb interval proximal to Nppa, or in a 1- to 2-centiMorgan (cM) interval distal to Nppa. In these congenic strain analyses, the Nppa and Fv1 loci, in addition to genes within about 1 cM of these loci, have been excluded as candidates for the Pctr2 locus. A relevant locus that may reside in this interval is Rep2; it is associated with the efficiency of repairing X-ray induced DNA damage sustained during the G2 phase of the mitotic cycle. The Pctr2 locus acts in a codominant fashion. F1 hybrids between resistant and susceptible congenic strains exhibit a reduced tumor incidence and a significant delay in the onset of tumorigenesis. Identification and eventual cloning of the Pctr2 locus may assist in the identification of genes involved in many types of cancer showing aberrations in human chromosome 1p36.

BALB/c.CBA/N mice carrying the defective Btk(xid) gene are resistant to pristane-induced plasmacytomagenesis.

The X-chromosome from the CBA/N mouse which carries the defective Bruton's tyrosine kinase (Btk) allele (Xxid) has been introgressively backcrossed onto the plasmacytoma (PCT) induction-susceptible BALB/cAN. Inbred BALB/c.CBA/N-xid/xid (C.CBA/N) mice raised and maintained in our conventional colony were given three 0.5 ml injections of pristane and were highly refractory to PCT induction. Only one PCT was found among 59 mice followed for > or =300 days. Twenty mice were examined at day 200 for foci of plasma cells in the oil granuloma. Ten mice had small foci of plasma cells, most of which were plasmacytotic, embedded in the inflammatory oil granuloma. In one there were multiple foci, but most of the mice had only one or two foci. F1 hybrid XxidY males derived from CBA/N females crossed to BALB/cAnPt were also resistant to PCT induction, while heterozygous and homozygous XY males were susceptible. C.CBA/N mice can develop extensive mucosal plasma cells as well as plasma cell accumulations in oil granuloma tissue, but the precursors of these plasma cells do not give rise to PCT in genetically susceptible hosts. The failure of C.CBA/N mice to develop PCT is probably due to the elimination of B cell clones that can be perpetuated by repeated exposure to thymus-independent type 2 antigens.

Construction of a BALB/c congenic mouse, C.C3H-Lpsd, that expresses the Lpsd allele: analysis of chromosome 4 markers surrounding the Lps gene.

Development of a congenic BALB/c mouse strain that contains a segment of chromosome 4 including the Lpsd allele of the lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ strain is presented. On the basis of LPS-induced spleen cell mitogenesis, macrophage tumor necrosis factor secretion, and tyrosine phosphorylation in vitro and lethality in galactosamine-sensitized mice in vivo, the C.C3H-Lpsd strain provides a model of LPS hyporesponsiveness that is comparable to that of the parental C3H/HeJ strain. Analysis of markers in this region indicates that length of the donor fragment is approximately 5.5 centimorgans. Thus, the C.C3H-Lpsd strain provides an important genetic tool for analysis of markers in this region and for examining functional effects of Lpsd expression on the BALB/c background.

A BALB/c congenic strain of mice that carries a genetic locus (Ityr) controlling resistance to intracellular parasites.

BALB/c.DBA/2 Idh-1b-Ityr-Pep-3b congenic mice were developed by introgressively backcrossing the Idh-1b and Pep-3b markers of DBA/2 mice onto the BALB/c pi mice. This introduced a 30-centimorgan chromosome 1 segment of DBA/2 chromatin that contained the Ityr gene. BALB/c.DBA/2 Idh-1b-Ityr-Pep-3b mice were resistant to in vivo infections by Salmonella typhimurium, Mycobacterium bovis, and Leishmania donovani.