Int. J . Cancer: 49, 224-228 (1991) Publication of the International Union Against Cancer Publication de I'Union Internationale Contre le Cancer 0 1991 Wiley-Liss, Inc.

DIFFERENTIAL SUSCEPTIBILITY OF BALBlc AND DBA12 CELLS TO PLASMACYTOMA INDUCTION IN RECIPROCAL CHIMERAS Santiago SILVA, Hiroyuki SUGIYAMA', Magdalena BABONITS, Francis WIENER and George KLEIN Department of Tumor Biology, Karolinska Institutet, Box 60 400, S-I04 01 Stockholm, Sweden.

Reciprocal chimeras were generated between BALB/c and DBN2 mice by inoculating newborn recipients of either strain with bone-marrow (BM) cells of the other through the peri- orbital vein. DBA/2 mice inoculated with the BALB/c with proven chimerism will be referred to as C-D, the reciprocal as D -4 . The BALB/c cells carried a Robertsonian 6;15 (Rb6; 15) chromosome marker to facilitate identification. The chimeric mice contained between 5% and 70% of donor cells when examined at 4 to 5 weeks of age. Six of 10 C-D devel- oped plasmacytomas (MPC) after 3 X 0.5 ml monthly pris- tane treatment (incidence 60%) and 8 of 25 (incidence 32%) after 2 to 3 X 0.5 ml pristane followed by Abelson virus (A- MuLV) infection. Seven of 15 W C developed MPC after pris- tane t reatment (incidence 47%) and 4 of 17 after pristane + A-MuLV (incidence 24%). All tumors that have arisen in both reciprocal chimeras originated from BALB/c cells independently of the degree of chimerism. All tumors contained an Iglrnyc translocation. Among the C+D chime- ras, 5 carried t(l2;IS) and I t(6;15) in the pristane-treated group, while 4 carried t( 12; I5), I t(6; 15) and 3 t(l5; 16) in the pristane + A-MuLV. Among the W C chimeras 6 carried t( 12; 15) and I t(6; 15) in the pristane-treated group, while 3 t(l2;IS) and I t(6;15) in the pristane + A-MuLV. No tumors developed in 18 pristane- and 22 pristane + A-MuLV-treated DBAlZ mice nor in I 5 pristane- and 17 pristane + A-MuLV- treated (BALB/c X DBA/2)F, mice. The data indicate that BALB/c and DBN2 cells differ in their propensity to trans- form into plasmacytoma in identical host environments after both pristane and pristane + A-MuLV treatment. They also show that the oil granuloma can support MPC development in either type of chimeric host.

Murine plasmacytomas (MPC) can be induced by intraper- itoneal (i.p.) injection of pristane oil. The predisposition to develop MPC is limited to a few strains, such as BALB/c and NZB. Other strains such as A/He, AKR, C3H, C57B1 and DBA/2 are resistant (R), (Potter, 1984). Susceptibility (S) var- ies among different BALB/c sublines ranging from 10% in BALBlcJ (Potter and Wax, 1981) to 61% in BALBkAnPt (Potter and Wax, 1983). F, hybrids between S and R strains are resistant to MPC induction.

The vast majority of MPC carry Iglmyc translocations char- acterized by the juxtaposition of c-myc sequences on chromo- some 15, to IgH sequences on chromosome 12 in the typical translocation or to IgLK on chromosome 6 or IgLX on chro- mosome 16 in the variant translocations (Ohno et al., 1979; Klein and Klein, 1985; Wiener et al., 1990).

Plasmacytoma cells can be detected in the ascitic fluid 3 months after the start of pristane treatment. MPC develops only after a latency period of 180 to 360 days however, with a mean latent period of 210 to 220 days. Tumor development is de- pendent on the presence of an oil granuloma (OG). Even es- tablished tumors are dependent on the presence of an OG to grow in syngeneic recipients after transplantation. The infec- tion of pristane-primed BALB/c mice with Abelson virus can reduce the latency period to 35 to 70 days post viral infection (Potter et al., 1973). We have found that Abelson virus infec- tion of BALB/c spleen cells in vitro makes them also suscep- tible to rapid MPC development in pristane-treated syngeneic recipients (Sugiyama et al., 1989).

Induction experiments on segregating BALB/c.DBA/2 crosses and congenic sub-lines have suggested that the DBA/2

strain carries at least three dominantly acting, unlinked resis- tance genes, not present in BALB/c (Potter, 1984; Potter and Wax, 1985). One of these genes resides on chromosome 4 (Potter et al., 19886).

The purpose of our present study was to examine whether the resistance of the DBM2 mice to MPC-induction was de- termined at the level of the PC precursor cell itself or at the level of the host. This was examined in reciprocal chimeras. BALB/c-derived cells were distinguished from DBN2 cells by the presence of a Robertsonian 6;15 chromosome marker. We found that MPCs could be induced in both reciprocal chimeras but were exclusively of BALB/c origin.

MATERIAL AND METHODS

Mice Six- to 8-week-old mice were used as bone-marrow (BM)

cell donors. BALB/c6; 15 were derived from BALB/cAnPt background, by introducing a Rb6; 15 fusion chromosome that arose spontaneously in our BALB/cJ colony. DBAR mice of our own inbred nucleus have been derived from founding pairs, originally obtained from the Jackson Laboratories. The mice were bred and kept in our department in conventional condi- tions.

Production of chimeras BM from donor mice were flushed with buffer salt solution

(BSS). Tubes containing the cell suspensions were maintained on ice. Cells assigned for inoculation were filtered through 20-pm-pore-size nylon gauze. Newborn recipients (less than 24 hr old) were injected through the peri-orbital route at days 1 , 2 and 3 with 2 x lo4 nucleated BM celldday in a volume of 30 pl with 30-gauge needles and tuberculin syringes. Chime- rism was assessed in 4-to-5-week-old mice, after weaning. Stimulated peritoneal lymphocytes were karyotyped as de- scribed by Silva et al. (1989). At least 20 metaphase plates were counted. Cells with the Rb6;15 marker were diagnosed as of BALB/c, normal karyotype as of DBAR origin. Only chi- meras carrying more than 10% of donor cells were used for the tumor induction experiments.

Plasmacytoma induction The C-+D and M C chimeric mice were subdivided in two

MPC induction groups. The 1st group received 0.5 ml pristane i.p. 3 times at monthly intervals. The 2nd group was injected monthly with either 2 X or 3 X 0.5 ml dose of pristane fol- lowed by A-MuLV (helper-free) infection i.p. 2 to 3 weeks later. The psi-2pAB4 cell line (a kind gift from Dr R. Risser) was used to obtain helper-free Abelson virus as described by Sugiyama et al. (1989). The pristane-treated chimeras were injected i.p. with 0.5 ml (2 X lo5 ffuiml) of filtered superna- tant.

'Present address: Saisekai Noe Hospital, 2-33 Imafukuhigashi 2-chome, Jbtb-Ku, Osaka 536, Japan.

Received: February 19, 1991 and in revised form May 13, 1991

SUSCEPTIBILITY OF BALB/c AND DBA/2 CELLS TO PLASMACYTOMA INDUCTION 225

Plasmacytoma diagnosis Each mouse that developed ascites was punctured with a

25-gauge needle attached to a 2-ml syringe. One mi of ascites was collected and diluted with BSS containing 5% fetal calf serum (FCS). Cytosmears were stained with May-Griinwald- Giemsa. The MPC diagnosis was assigned when more than 10 large plasmacytoid cellslslide were found with intense blue- stained cytoplasm and eccentric non-picnotic nucleus with a clear perinuclear zone (hof). Diagnosis was further confirmed by identifying an MPC-associated typical or variant transloca- tion after one i.p. transplantation into primed syngeneic mice. The cytoplasmic Ig class was determined in parallel on cy- tosmears by indirect immunofluorescence, using affinity- purified goat anti-mouse p, ?, 6 and (Y chain antibodies (Sigma, St. Louis, MO) and FITC-conjugated rabbit anti-goat Ig antibody (Hyland, Glendale, CA).

The BALBIc vs. DBN2 origin of the tumor was based on the chromosome examination for the Rb6;15 marker. Chromo- somal preparations were made from peritoneal fluid collected from ascites-carrying mice as described by Silva et al. (1989).

RESULTS

Chimerism Eighty-nine newborn DBAl2 were injected through the peri-

orbital vein with BALBIc-derived BM cell suspensions in

70

60

50

40

30

20

10

0

70

60

50

40

30

20

10

0

PRISTANE

+ +

+ + +

+

C+D chimeras. Fifty mice survived and were assayed for chimerism after weaning. Thirty-five C-D chimeras con- tained between 10% and 65% donor cells (Fig. la) . Eighty BALBIc newborn mice were injected with BM cells from DBM2 donors in the reciprocal D - C chimeras. Fifty-one were weaned and 32 of them carried between 10% and 70% donor cells (Fig. l b ) . Mice containing less than 10% donor cells were sampled together with unmanipulated mice and used as control. Both C-D and D+C chimeras were randomly subdivided in two MPC-induction groups (Fig. la&).

Histopathology The second pristane injection was usually followed by a

large accumulation of ascites in C+D mice. The early ascites contained only very few cells as a rule. The peritoneal fluid was repeatedly drained, to keep the mice alive. Cytosmears prepared after the second pristane injection showed predomi- nantly macrophages with occasional groups of monocytes and polymorphonuclear leukocytes. Whenever a small fraction of PC cells appeared to be present, serial smears were prepared every 2nd week. There was no systematic difference between early- and late-appearing tumors with regard to the level of chimerism (Fig. la ,b) .

Plasmacytomas In C-+D chimeras treated only with pristane, a few isolated

plasmacytoma cells were observed within 5 to 6 months of the

0 29

PRISTANE + A-MuLV

0 0 0 115

2 1 117 160

I I

a

PRISTANE + A-MuLV

b

0 125

-152 0 130 k , : ; , , ; 152

0 100 200 300

L A T E N C Y (DAYS1

RGURE 1 - Donor-cell persistence (%) at 4 to 5 weeks of age in C+D and W C chimeras and tumor latency. (0) Chimeric mice treated with pristane (3 X 0.5 ml) that did not develop any MPC after an observation period of 350 days; (+ ) MPC development; (0) chimeric mice treated with 2 X 0.5 ml of pristane + A-MuLV without MPC development; (0) mice carrying tumors; (a) mice treated with 3 X 0.5 mi pristane + A-MuLV without MPC development and (w) with MPC development. The latency post-virus-infection is given under the corre- sponding tumors.

226 SILVA ET AL

'O

60

50

uo

30

20

10

TABLE I - PLASMACYTOMA INCIDENCE IN C+D AND D-C CHIMERAS

Plasmacytoma incidence

TEPC ABPC

Donor Host Donor Host

Chimeric group

T E P C : PP a - C + D =

- D + C = M

- Control=

- - -

C-D 6/10 (60%) - 8/25 (32%) - M C - 7/15 (47%) - 4/17 (24%)

70

60

50

uo

Control BALB/c - 12120 (60%) - 7125 (28%)

- C -> D

- D -> C = M u

- Control =++- -

DBA/2 - 0/18 (0%) - 0122 (0%) CDF 1 - 0/15 (0%) _. 0/17 (0%)

30

20

10

-

-

-

I I

0 100 200 300

0

100 200 300

L A T E N C Y ( D A Y S )

FIGURE 2 - Cumulative % of plasmacytoma incidence in C+D and I k C chimeras following pristane (TEPC) and pristane + A-MuLV (ABPC) treatment. Comparison with TEPCs and ABPCs induced in conventional BALB/c mice.

first pristane injection. Tumors developed only after latency periods of more than 6 months post-pristane. Six MPC of donor origin developed in the 10 C+D chimeras treated only with pristane (TEPC) (incidence = 60%) (Table I , Fig. 2a). The tumors developed after latencies ranging between 165 and 244 days (Table 11). Altogether 25 C+D chimeras were treated with pristane and infected with Abelson virus (helper-free) and 8 of them (incidence = 32%) developed MPC (Fig. 2b). Two mice in this group (163-10 and 163-1) that received 2 X 0.5 ml pristane followed by A-MuLV infection developed MPC less than 4 months after the 1st pristane injection, and only 21 and 29 days respectively post viral infection. A 3rd C+D chimeric mouse (119-10) also developed MPC shortly (36 days) after virus infection. Nevertheless, 5 out of 8 ABPC developed in C+D chimeras after relatively long post-pristane and post- virus latencies (Table 11, Fig. I&). In D+C chimeras, MPC developed 4 months after the 1st pristane injection in mice

treated only with pristane. Seven of the 15 (47%) treated mice developed TEPC with latency periods ranging between 117 and 278 days post-pristane, (Table 11). Among M C chimeras treated with pristane + A-MuLV, MPC were observed only 4 to 5 months post virus infection. Four MPC were registered among the 17 D-.C chimeras treated with pristane + A- MuLV (incidence = 24%). All ABPC developed in D+C mice after long post-viral latencies (Table 11, Fig. lb).

Lymp hosarcomas Three lymphosarcomas (LS) developed in C+D chimeras

treated with pristane + A-MuLV. One of them was derived from host cells, the other 2 were of donor origin. Chimeric mouse 163-1 developed both a LS (ABLS-Ch-163-1) and a MPC (ABPC-Ch-163-1). During early in vivo passage the LS overgrew the ABPC (Table 111).

Macrophagelhistiocytic tumors Four MQIH type tumors developed in C+D chimeras. Two

of them (ABMQ-Ch-129-3- and 131-2) were derived from host cells in chimeras that received pristane + A-MuLV. A 3rd (TEMQ-Ch-144-30) was of donor type and developed after a latency of 344 days in a mouse treated with pristane only. The 4th tumor in this group (ABMQ-Ch-132-0) was a mixture of host + donor cells (Table 111).

Leukemias

chimeras. The tumors originated from host cells (Table 111).

Cytogenetic analysis All MPC originated in BALB/c cells, in both reciprocal

chimeric groups. This means that the MPC in the C+D group were all donor type, whereas in the I k C group they were all host type. The majority of MPC were diploid at an early stage of MPC genesis. They became near-diploid or tetraploid in late stages of tumor development. The typical rcpt(12;15) was the predominant translocation observed in both chimeric groups. Five of 6 TEPC in C+D chimeras (TEPC-Ch-144-1, 144-3, 144-20, 144-21 and 162-1) and 6 of 7 TEPC in D-tC chimeras (TEPC-Ch-176-10, 180-3, 181-10, 181-11, 219-1 and 220-1) carried t(12;15), while TEPC-Ch-144-0 and TEPC-Ch-181-2 carried t(6; 15) variant translocation. The corresponding figures were 4 out of 6 ABPCs in C+D (ABPC-Ch-119-2, 119-10, 152-0 and 152-3) and 3 of 4 ABPC in M C (ABPC-Ch-265-3, 266-2 and 266-10) carrying a t(12;15). Two tumors (ABPC- Ch-129-0 in C+D and ABPC-Ch-285-2 in M C ) had variant t(6;15) translocations. Three ABPCs (ABPC-Ch-119-3, 163-1 and 163-10) in C+D chimeras carried a t(15;16) variant trans- location. Molecular analysis of the line established from ABPC-Ch- 163- 10 confirmed the postulated lambddmyc trans- location (data not shown). In addition to Iglmyc translocations, several MPCs had an extra copy of Rb6;15. In tumors TEPC- Ch-144-3 and ABPC-Ch-152-3 the translocated Rb(6;de115)12 element was duplicated. In ABPC-Ch-163-1 and 163-10 the translocated Rb(6;del15)16 was duplicated. In contrast, in 5 other MPCs (TEPC-Ch-144-21, 181-1, 181-11, 219-1 and ABPC-Ch- 1 19-3) the non-translocated Rb6; 15 chromosome was duplicated. An extra copy of chromosome 11 was also found frequently in both chimeric groups.

Immunoglobulin production The results of the Ig analysis are presented in Table 11. The

tumors were predominantly IgAK or IgGK. The tumors TEPC- CH-144-1 and 144-21 produced both IgA + M and IgA + G respectively. These tumors became non-producers after in vivo passage. Two primary ABPCs (ABPC-Ch-152-0 and 152-3) in the C+D group were non-producers.

Two leukemias of myeloid type were observed in C+D

SUSCEPTIBILITY OF BALB/c A N D DBAR CELLS TO PLASMACYTOMA INDUCTION

TABLE I1 - LATENCY AND CYTOGENETIC FEATURES OF PLASMACYTOMAS INDUCED IN C+D AND I k C CHIMERAS

227

agents %' PP2 PV3 Ig Origin Cytogenetics4 Induction Chimerism Latency period Tumor name Chimera P U P

C-+D TEPC-Ch- 144-21 TEPC-Ch- 144-20 TEPC-Ch- 162-1 TEPC-Ch-144-0 TEPC-Ch- 144-1 TEPC-Ch- 144-3 ABPC-Ch- 163-10 ABPC-Ch-163-1 ABPC-Ch- 129-0 ABPC-Ch- 152-3 ABPC-Ch-119-10 ABPC-Ch- 152-0 ABPC-Ch-119-2 ABPC-Ch-119-3

C+D TEPC-Ch- 18 1-1 1 TEPC-Ch-176-10 TEPC-Ch-181-10 TEF'C-Ch-219-1 TEPC-Ch-220- 1 TEPC-Ch- 180-3 TEPC-Ch- 18 1-2 ABPC-Ch-285-2 ABPC-Ch-266- 10 ABPC-Ch-266-2 ABPC-Ch-265-3

PPP5 PPP PPP PPP PPP PPP PPV6 PPV PPV PPV PPPV6 PPV PPPV PPPV

PPP PPP PPP PPP PPP PPP PPP PPV PPV PPV PPV

45 I65 25 176 15 198 55 214 65 214 25 244 35 106 60 114 35 178 40 215 15 242 30 260 10 306 15 306

35 117 10 147 15 175 10 213 15 222 10 223 20 218 10 213 35 259 15 264 15 286

AKG, A, G ,

A N , A,

- - - - - -

21 A, 29 N.D.

117 G, 117 N.P. 36 G,

160 N.P. 96 A, 96 A,

- G K

G, A,

-

- - - - N.D.

152 A, 125 G, 130 N.D. 152 A,

- N.D.

DONOR DONOR DONOR DONOR DONOR DONOR DONOR DONOR DONOR DONOR DONOR DONOR DONOR DONOR

HOST HOST HOST HOST HOST HOST HOST HOST HOST HOST HOST

t(12;15), + (Rb6;15), M(Rb3;9) t(12;15), + 1, t(4;7) t( 12; 15) inv(6: 15) t( 12; 15) ' t(12;15), + (Rb6:de115)12 t( 15; 16), + (Rb6;dellS j16, + (Rbl1;ll) t(15;16), +(Rb6;de115)16 inv(6;15), + 18 t(12;15), +(Rb6;de115)12 + 11 t(12:15) t(i2;15j, + 11 t(12;15), + 11 t(ly;16), +(Rb6;15), +11

t(12;15), +(Rb6;15) t( 12; 15) t(12;15), + 11 t(12;15). +(Rb6:15) t( 12; 15) t( 12; 15) inv(6;15), +(Rb6;15) inv(6;15) t(12;15). + 11 ti 12; 15 j ' t(12;15), + 12

'Chimerism at weaning.-'Post 1st pristane injection.-'Post viral infe~tion.-~Numerical and structural aberrations.-'3 x 0.5 ml pri~tane.-~2 X ~ 3 x 0.5 ml + A-MuLV.

TABLE I11 - LATENCY AND CYTOGENETIC FEATURES OF 9 NON-PC TUMORS DEVELOPED AFTER PRISTANE OR AFTER PRISTANE + A-MuLV TREATMENT IN C-D CHIMERAS

. "..._. TY pe Designation

Latency periods

PV* Induction c ~ ~ ~ ~ m (days) Origin

T,,"WW

agents at weaning p p ~ Chromosome aberrations

60% 113 28 (d)4 Diploid ABLS-Ch- 129-1 ppv3 DDV 15% 264 206 (d) Diploid

Lymphosarcoma ABLS-Ch-163- I

ABLS-Ch- 15 1-2 bpv 20% 277 177 (h? Diploid 25 % 105 51 (h) Diploid 15% 116 65 (h) Diploid 15% 267 213 (d/h) Tetraploid/Near-diploid6 25% 344 - (d) Near-diploid +7, +MI

Macrophage- ABM@-Ch-131-2 PPV histiocytic ABM@-Ch-129-3 PPV

ABMQ-Ch-132-0 PPV TEM@-Ch-144-30 ppp

20% 116 55 (h) Diploid 15% 242 32 (h) Hvuerdidoid +6. +7. +15, +18

Myeloid ABML-Ch- 129-2 PPV ABML-Ch-I 19-10' PDDV

'After 1 st pristane injection.-'PostviraI infection.-'This LS developed simultaneously with ABK-Ch-163-1 in the chimeric mi~e . -~Host . -~Donor . -~A single tumor type was histologically identified, but both donor and host cells showed cytogenetic changes.-'This host-derived myeloid tnmor grew parallel with the donor-derived ABF'C-Ch- 119.10 in the same chimeric mouse.

Tumorigenicity Ascites from primary-tumor-bearing mice was injected i.p.

into primed and unprimed conventional BALB/c, CDF, and DBA/2 recipients. All the tumors grew in primed BALB/c and CDF, mice, while none of them grew in primed DBN2. None of the passaged tumors grew in unprimed mice (data not shown).

DISCUSSION

Only BALB/c cells gave rise to MPC in these experiments, whether they were of host or of donor origin. In the C+D chimeras they were all of donor origin, whereas in D+C chi- meras they were all of host origin. This suggests that at least part of the difference in the susceptibility of BALB/c and DBM2 mice to MPC induction is determined at the level of the precursor cell itself. It also follows that the oil granuloma of

both the BALB/c and the DBA/2 mice can support PC devel- opment. This is stated with the reservation that both types of host granulomas can be expected to contain an unknown num- ber of donor cells in the chimeric animals.

The question arises whether B-cells of DBA/2 vs. BALB/c origin may differ in their propensity for Iglmyc translocations. Potter et al. (1988a,b) argued that susceptibility and resistance to plasmacytomagenesis may be due in part to genes that in- fluence the efficiency of chromatin repair. Using C.D congenic lines, they have found that partially resistant C.D2Fv-l(N19) mice carried a resistance gene in the telomeric region of chro- mosome 4. They also had a gene for more efficient chromatin repair, as compared with BALB/cAn mice. The same authors suggested that the recessive MPC-susceptibility gene in S BALB/cAn mice may increase the likelihood of DNA damage that would predispose for chromosomal translocations.

Chimeric mice provide an opportunity for the critical exam- ination of the question whether cells of different genotypes

228 SILVA ET AL.

differ in their proneness to generate an Iglmyc translocation and whether that difference, if detected at the cell level, is fixed at the level of chromosome 15 itself or at some other level.

14054-15 from the US National Cancer Institute and by the Swedish Cancer Society. S.S. is a recipient of fellowships from the Cancer Research Institute, New York and the Concern Foundation, Los Angeles, CA. H.S. was supported by fellow- ships from the Swedish Cancer Society and the Cancer Re- search Institute, N.Y.

ACKNOWLEDGEMENTS

This investigation was supported by PHS grant 5 RO1 CA

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