Santiago Silva

Epstein‐Barr virus nuclear antigen‐1 and Myc cooperate in lymphomagenesis

The lymphomagenic action of myc genes in conjunction with Epstein‐Barr virus nuclear antigen‐1 (EBNA‐1) have been examined using transgenic mice in several separate tests. Synergy between Myc and EBNA‐1 in lymphomagenesis was revealed in a cross breed study where co‐expression of transgenic myc and EBNA‐1 led to a tumor latency period reduced significantly in some crosses. In the resulting bitransgenic tumors, expression of the Eμ‐myc genes was not affected by EBNA‐1 expression. MoMLV was utilized as a transposon tag to activate cellular oncogenes by infection of EμEBNA‐1 mice. Rearrangement at the c‐myc locus in B cell tumors from these mice again suggests a cooperative action between myc and EBNA‐1. Tumors arising in EμEBNA‐1 mice typically showed a trisomy of chromosome 15, upon which the c‐myc locus resides. Bitransgenic tumors (EBNA‐1/c‐myc) did not show trisomy 15. This raises the possibility that amplification of c‐myc is factorial in the selection of trisomy 15 in these tumors. These data indicate that myc and EBNA‐1 act cooperatively and are not redundant in lymphomagenesis. Expression of EBNA‐1 by EBV may provide a selection pressure in addition to translocation of the c‐myc locus in the genesis of endemic Burkitts lymphoma (BL).

Location of Myc, Igh, and Igk on Robertsonian fusion chromosomes is inconsequential for Myc translocations and plasmacytoma development in mice, but Rb(6.15)-carrying tumors prefer Igk-Myc inversions over translocations.

The location of the Myc and immunoglobulin (Ig) loci on metacentric Robertsonian (Rb) fusion chromosomes may affect the development of mouse plasmacytomas (Pcts) by changing the probability with which chromosomal Myc–Ig translocations occur. To test this hypothesis, we induced Pcts in BALB/c (C) mice that carried Rb(4.12) and/or Rb(6.15) chromosomes. The Rb mice developed Pcts (n = 198) with similar onset and incidence to that in the inbred C mice. Karyotyping of 70 Rb–carrying Pcts demonstrated that in these tumors, just as in their counterparts in inbred C mice, the Igh heavy‐chain locus was translocated with Myc more often than was the Igk light‐chain locus. Pcts harboring Igh or Igk on normal and Rb chromosomes showed no bias toward either in generating Myc translocations. These findings indicated that the location of Myc, Igh, and Igk on normal or Rb chromosomes is inconsequential for Myc translocation and Pct development. In contrast, in Rb(6.15) mice, in which chromosomal inversions competed with chromosomal translocations for Igk–Myc juxtapositions, the former occurred more frequently than the latter in the resulting Pcts. This suggested that spatial proximity of Igk and Myc on the same chromosome facilitates the rearrangement of these loci. Myc translocation–dependent mouse Pct may provide a good model system for furthering our understanding of the relationship of higher‐order genome organization in the interphase nucleus, origin of chromosomal translocations, and development of cancer. Published 2005 Wiley‐Liss, Inc.

Spontaneous development of plasmacytomas in a selected subline of BALB/cJ mice

Sixty per cent of BALB/cAnPt mice injected intraperitoneally (i.p.) with tetramethylpentadecane (pristane) develop plasmacytomas (PCs), whereas less than 10% of BALB/cJ develop such tumours. Most other mouse strains are completely resistant. Resistance is dominant over susceptibility in F1 hybrids between BALB/cAnPt and the resistant non-BALB/c strains, suggesting that susceptibility may be due to some genetic defect. (BALB/cAnPtxBALB/cJ)F1 hybrids have a PC incidence of 36-42%. Previously, BALB/cJ has been shown to harbour at least one resistance gene (Potter et al., Genomics 1988, Vol. 2, pp. 257-262). On the assumption that BALB/cJ may contain a segregating resistance gene, we cross BALB/cJ females with pristane-pretreated BALB/cJ males that were found to be carrying PC cells intraperitoneally 5-7 months after pristane treatment. After two selective crosses, 62% of the BALB/cJ subline BALB/cM2/22 developed PC after pristane and 52% after pristane followed by Abelson virus, while unselected controls had an incidence of 11% and 0%, respectively. Moreover, six spontaneous plasmacytomas developed in untreated females of the selected colony. Five of these carried T(12; 15) (F2; D2/3) translocations. The sixth had a T(1; 10) (G; C1) translocation and an interstitial duplication of segment (C1/E3) on one chromosome 5. It may be concluded that pristane treatment is not a prerequisite for the induction of the PC associated Ig/myc translocations.

Plasmacytoma Induction in Specific Pathogen-Free (SPF) bcl-2 Transgenic BALB/c Mice

Germ-free (GF) and specific pathogen free (SPF) BALB/c mice are refractory to plasmacytoma induction by pristane (McIntire and Princler, 1969, Byrd et al 1991). It was therefore suggested that MPC development may depend on antigenic stimulation. If so, it may conceivably act by preventing the apoptotic elimination of tumor precursor cells. We have tested this idea by elevating the apoptotic threshold by the introduction of a bcl-2 transgene. We have found that MPCs could be induced by pristane oil in transgene carrying SPF mice. An E mu activated bcl-2 transgene was introduced into SPF BALB/c mice. The mice were used after two backcrosses (BC-2). Pristane oil treatment was started at 4 to 6 weeks of age (3 x 0.3 ml via i.p. at monthly intervals). For each transgene carrier a transgene negative littermate was used as control. Fifteen of 24 (63%) transgene carriers developed plasmacytomas after latency periods between 67 and 146 days (mean = 112 +/- 30 days) after the first pristane injection. Five additional transgene carriers developed lymphoma (3 cases) or mixed MPC and lymphoma (2 cases). In contrast, no tumors developed in 16 transgene negative littermates that were kept > 300 days under observation. Karyotyping showed that 10/15 (66%) of the MPCs carried a T(12;15) translocation, 4/15 (27%) carried both T(12;15) and T(6;15) translocations in the same metaphase plate, and 1/15 (7%) was translocation free. A T(12;15) translocation was also detected in one of the 2 mice with mixed tumor type. Pristane treated bcl-2 transgenic C57B1/6 mice remained tumor free, although T(12;15) translocation carrying cells were found in the peritoneal fluid of 4/20 mice 176 days after pristane.

Ig/myc translocations of the plasmacytoma-prone BALB/c strain occur independently of the genetic and parental origin of the affected chromosomes 6, 12, and 15.

Virtually all murine plasmacytomas (MPCs) carry chromosomal translocations that juxtapose myc on chromosome 15 (chr 15) to one of the three immunoglobulin loci carrying chr 6, 12, or 16. Only some mouse strains are susceptible to MPC induction, however, with BALB/c as the outstanding example. Most other strains are resistant. Our earlier studies with reciprocal BALB/c→DBA/2 chimeras suggested that part of this susceptibility is determined at the level of the target cell itself (DBA/2 is MPC resistant). The probability of the lg/myc translocation is one of the possibly relevant variables. Because MPC resistance is dominant over susceptibility, it is conceivable that the translocations prevail due to some deficiency of the lg rearrangement or lg‐associated repair mechanisms in BALB/c cells. This could be determined at the level of the chromosomes that participate in the translocation or by genes on other chromosomes. Here, we show that the substitution of the BALB/c‐derived chr 12, 6, and 15, which carry lgH, K, and myc, respectively, with their homologs derived from MPC‐resistant mice, did not affect MPC susceptibility. The use of Robertsonian 4.12 and 6.15 chromosomes in this study has also provided us with the opportunity to assess the parental derivation of the chromosomes participating in the translocation. In contrast to the human chronic myeloid leukemia (CML)‐associated BCR/ABL fusion transcript, where a strong bias was claimed that was attributed to imprinting, we have found that the parental chromosomes were randomly involved in the translocation. We have also shown that the translocations could be of uniparental or biparental origin. Genes Chromosom Cancer 17:179–184 (1996).

Hypersomy of chromosome 15 with retrovirally rearranged c-myc, loss of germline c-myc and IgK/c-myc juxtaposition in a macrophage-monocytic tumour line

From a lymphoid tumour induced by 7,12-dimethylbenz-[a]-anthracene (DMBA) + methyl-N-nitrose-N-urea (MNU) in an [AKR Rb(6.15) x CBAT6T6]F1 mouse, a macrophage- monocyte line (KT-10) was isolated. Following ethyl methanesulfonate (EMS) treatment, a bromodeoxyuridine (BUdR) resistant subline was selected. Serial propagation of this line in vitro in the presence of BUdR (28 months) with periodic cytogenetic and molecular examinations, has led to the definition of four successive stages. During stage I, the cells were trisomic for chromosome 15. They contained Rb(6.15) and Rb(del6.15) of AKR and T(14;15) of CBA origin. Southern blotting showed the presence of both germline (G) and rearranged (R) c-myc. At stage II, Rb(del6.15) has duplicated. Both Rb(6.15) and T(14;15) persisted together with G-myc and R-myc. In stage III, the CBA-derived T(14;15) was lost, in parallel with G-myc. At this stage, a Dic.In(6.15) was detected. One of its arms was cytogenetically identical with the long arm of In(6.15) in the variant IgK/myc translocations. This chromosome carried R-myc and IgK in juxtaposition, as indicated by comigration on pulsed field electrophoresis (PFGE). At stage IV, the R-myc carrying AKR-derived chromosome 15s were present in six copies. Possible relationships between the increasing R/G myc ratio and changed growth characteristics in vivo and in vitro are discussed.

An Exceptional Mouse Plasmacytoma with a New Kappa/N-myc [T(6; 12) (C1; B)] Translocation Expresses N-myc But Not c-myc

Mouse plasmacytomas (MPC) carry one of three reciprocal translocations that juxtapose c-myc to one of the three immunoglobulin (Ig) loci. Here we describe an exceptional MPC, induced by pristane oil and Abelson (A-MuLV) virus. It does not carry any of the three c-myc/Ig translocations, but contains a previously unknown reciprocal T(6;12) translocation affecting the bands known to carry the IgK (6C/1) and N-myc (12B) loci, respectively. Northern blot analysis showed high N-myc but no c-myc expression. This is consistent with the constitutive activation of N-myc by a juxtaposition of the IgK and N-myc loci. Reciprocal translocation in B-cell derived tumors are believed to involve the Ig loci by the action of some enzyme that participates in the physiological rearrangement of the Ig loci. Only transcriptionally active chromatin regions are accessible to such recombinases (Alt et al. 1987). N-myc is not expressed in B-cells, but it is transcriptionally active during the early pro- and pre-B cell stage, whereafter it and the surrounding chromatin region becomes inactive (Smith et al. 1992). It is therefore most likely that the N-myc/Kappa translocation has arisen at an early stage of B-cell differentiation. This would imply that the myc/Ig translocations do not block B-cell differentiation. They also reaffirm the functional equivalence of N- and c-myc in relation to B-cell carcinogenesis, as shown by our previous work on tumor induction in N-myc transgenic mice (Wang et al. 1992).

Plasmacytoma Induction in BALB/c6;15 → DBA/2 Chimeras

Murine plasmacytomas (MPC) can be induced by intraperitoneal injection of 2, 6, 10,14 tetramethylpentadecane (pristane) into inbred BALB/c mice. Susceptibility to MPC development under similar regimen of pristane, range between 10 % in BALB/cJ (Potter and Wax, 1981), and 61 % in BALB/cAnPt with a mean latent period of 210–220 days (Potter and Wax, 1983). Other inbred strains such as A/He, AKR, C3H, C57B1 and DBA/2 develop very rarely MPC and had been considered as resistant to MPC induction, (Potter, 1984). The F1 hybrids between BALB/c and resistant parental strains, are also resistant to MPC induction, suggesting that resistance is under the control of genes that express dominancy, and susceptibility may represent the recesive allele, (Potter, 1984). The genes controlling susceptibility vs resistance have not been defined yet.

Mouse plasmacytoma associated (MPC) T(15;16) translocation occurs repeatedly in new MPC induction system

I would like to introduce my presentation with two slides published II years ago (Ohno et al. 1979). They illustrate the MPC associated 12:15 and 6:15 translocations. I do not intend to describe how successfully the whole story developed in the hands of molecular biologists. From the cytogenetic point of view, however, one intriguing question has remained unsolved (illustrated in Fig. 1).

Strain-Related Cellular Mechanisms as a Determinant for Susceptibility and Resistance to PC Induction

Plasmacytomas (PCs) can be induced in the susceptible BALB/c strain by intraperitoneal (i.p.) injection of pristane oil. They regularly carry an immunoglobulin (Ig)/myc translocation. The post- pristane latency period is usually more than 120 days. The minimal latency period can be shortened to one third by an injection of Abelson murine leukemia virus (A-MuLV), a potent pre-B lymphoma-inducing agent. The notion of co-operative interaction between deregulated myc and v-abl has been supported by our previous result that A-MuLV can rapidly induce translocation-free PCs in Eµ,-C- or N-myc transgenic mice at nearly 100% with or without pristane (Sugiyama et al., 1990; Wang et al., 1992). We recently found that rapid PC induction by A-MuLV occurs only in conjunction with deregulated myc, and by comparison of v-abl and a c-abl mutant, △XB, that the activity of the virus to induce pre-B lymphomas is related to its high titer presumably achieved by gag sequences (Sugiyama et al., in press). We further report here that the lymphoma-inducing activity of A-MuLV in N-myc mice is related to the upregulation of endogenous c-myc in contrast to the downregulation in rapidly induced PCs. We also show that strain-related susceptibility and resistance to PC induction is at least partly determined by cellular mechanisms other than the deregulation of myc.