J S Wax's research while affiliated with National Institute of Allergy and Infectious Diseases and other places

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.

Continuous indomethacin (INDO) administration in the drinking water (10 to 20 microg/mL) profoundly inhibited plasmacytoma (PCT) development initiated by three 0.2- or 0.5-mL intraperitoneal (i.p.) injections of pristane in hypersusceptible BALB/c.DBA/2-Idh1-Pep3 congenic mice. The most effective inhibitions were obtained with continuous INDO treatment. When treatment was delayed until 50 to 60 days after the first pristane injection, there was approximately a 50% reduction in PCT incidence. The primary action of pristane is the induction of a chronic inflammation in the peritoneal connective tissues and the formation of a microenvironment where PCTs develop. INDO, a powerful inhibitor of prostaglandin synthases (cyclooxygenases 1 and 2), did not inhibit the formation of mesenteric oil granuloma nor the appearance of cells in this chronic inflammatory tissue carrying c-myc illegitimately joined to an Ig heavy chain switch region, ie, the t(12;15) chromosomal translocation. INDO inhibited PCT induction by the i.p. implantation of 21 x 2 mm polycarbonate discs. These solid objects predominantly induce the formation of a patchy fibroplastic tissue on contacting peritoneal surfaces. These and previous data indicate that indomethacin inhibits an intermediate stage in PCT development after the arrival of cells bearing the T(12;15) translocation in the oil granuloma and before these cells acquire transplantability to a pristane-conditioned host. The biological mechanism that explains how INDO inhibits PCT development is not yet established but appears to result from decreased production of prostaglandins in chronic inflammatory tissues (oil granuloma, fibroplasia), suggesting that prostaglandins play an active role in oil and solid plastic induced PCT formation.

Continuous indomethacin (INDO) administration in the drinking water (10 to 20 μg/mL) profoundly inhibited plasmacytoma (PCT) development initiated by three 0.2- or 0.5-mL intraperitoneal (i.p.) injections of pristane in hypersusceptible BALB/c.DBA/2-Idh1-Pep3 congenic mice. The most effective inhibitions were obtained with continuous INDO treatment. When treatment was delayed until 50 to 60 days after the first pristane injection, there was approximately a 50% reduction in PCT incidence. The primary action of pristane is the induction of a chronic inflammation in the peritoneal connective tissues and the formation of a microenvironment where PCTs develop. INDO, a powerful inhibitor of prostaglandin synthases (cyclooxygenases 1 and 2), did not inhibit the formation of mesenteric oil granuloma nor the appearance of cells in this chronic inflammatory tissue carrying c-myc illegitimately joined to an Ig heavy chain switch region, ie, the t(12; 15) chromosomal translocation. INDO inhibited PCT induction by the i.p. implantation of 21 × 2 mm polycarbonate discs. These solid objects predominantly induce the formation of a patchy fibroplastic tissue on contacting peritoneal surfaces. These and previous data indicate that indomethacin inhibits an intermediate stage in PCT development after the arrival of cells bearing the T(12; 15) translocation in the oil granuloma and before these cells acquire transplantability to a pristane-conditioned host. The biological mechanism that explains how INDO inhibits PCT development is not yet established but appears to result from decreased production of prostaglandins in chronic inflammatory tissues (oil granuloma, fibroplasia), suggesting that prostaglandins play an active role in oil and solid plastic induced PCT formation.

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.

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.

Although plasmacytomas can develop spontaneously in mice, the more common origin requires inducing agents. In the system which we will describe, plasmacytomagenesis is initiated by the intraperitoneal injection of paraffin oils such as the alkane pristane (2,6,10,14-tetramethylpentadecane) (Anderson and Potter 1969). These tumors can also be induced by the intraperitoneal implantation of solid plastic materials as was originally discovered by Merwin and Algire (see Merwin et al. 1963). An important characteristic of plasmacytoma induction in mice is that it only works in certain inbred strains such as BALB/cAn in which the expected average incidence is ∼ 60%, or NZB where between 30 and 40% of the mice develop these tumors; most other inbred strains do not develop these tumors (Morse et al. 1980). Plasmacytomas have been studied cytogentically by Francis Wiener and Shinsuke Ohno, and over 95% of them have reciprocal chromosomal translocations involving immunoglobulin genes (chromosomes 12 and 6 and 16) and c-myc (chromosome 15) or loci near c-myc such as Pvt-1 (Ohno et al. 1979,1984). These translocations cause deregulation of c-mvc transcription (Mushinski 1988). Deregulation of c-myc cooperates with other oncogenic mutations to transform certain B-lymphocytes but does not appear to be sufficient by itself to cause transformation (see Potter 1990). C-myc deregulation is consistently found in spontaneous rat immunocytomas (plasma cell tumors) and in Burkitt’s lymphoma in man (see Potter 1990).

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.

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.

A retroviral vector, RIM, containing murine c-myc under the control of immunoglobulin heavy-chain gene promoter and enhancer elements and v-Ha-ras driven by a Moloney murine leukemia virus long terminal repeat induced IgM-secreting plasmacytomas in 28% of adult and 83% of 3-week-old pristane-conditioned mice with mean latency periods of 60-70 days. In contrast, the same vector only harboring c-myc or v-Ha-ras was virtually ineffective. RIM-induced plasmacytomas expressed retroviral myc and ras genes while their endogenous c-myc alleles were unrearranged and transcriptionally inactive. These plasmacytomas were clonal as each possessed a unique immunoglobulin heavy-chain joining region rearrangement and a single recombinant provirus. Moloney murine leukemia helper virus did not play an obligatory role in tumorigenesis since insertions of Moloney murine leukemia proviruses were found in only 6 of 24 plasmacytomas induced in adult mice. Taken together, these findings support the view that the v-Ha-ras oncogene can cooperate with an activated myc gene in pristane plasmacytomagenesis.

BALB/cAn mice are susceptible to the induction of plasmacytomas (PCTs) by the intraperitoneal injection of pristane. An average of 60% of the mice inoculated with pristane develop tumors with a mean latent period of 220 days (Potter et al. 1984). DBA/2 mice are resistant to PCT formation (0% incidence by Day 220). Resistance in DBA/2 is a dominant trait as (BALB/cAnPt x DBA/2N) F1 mice are solidly resistant to the plasmacytomagenic process. The incidence of PCTs in F2 (<2%; unpublished data) and backcross progeny (11%; Potter et al. 1984) between BALB/cAnPt (PCTS) and DBA/2N (PCTR) progenitors is suggestive of a model for 3 recessive genes for resistance to PCT formation. Over 95% of the tumors which develop in BALB/cAn mice carry either rcpt (6;15) or rcpt (12;15) translocations involving an Ig locus and c-myc directly or indirectly. Attempts have been made to bypass some of the earlier genetic events in the pathway to PCT formation through the introduction of specific oncogenes via retroviral vectors. Vectors carrying v-abl (Potter et al. 1973) or combinations of v-raf and v-myc (Potter et al. 1987) have produced PCTs in at least 20% of the pristane-primed BALB/cAn mice inoculated with these retroviral constructs by as early as day 80 post pristane.