Int. J. Cancer: 41, 738-743 (1988) Publication of the International Union Against Cancer Publication de I’Union Internationale Contre le Cancer 0 1988 Alan R. Liss, Inc.

FURTHER STUDIES ON CHROMOSOME 15 TRISOMY IN MURINE T-CELL LYMPHOMAS: MAPPING OF THE RELEVANT CHROMOSOME SEGMENT Santiago SILVA, Magda BABONITS, Francis WIENER and George KLEIN Department of Tumor Biology, Karolinska Institutet, Box 60400, S-I04 01 Stockholm, Sweden.

Trisomy 15 is the most common chromosomal aberration in murine T-cell lymphomas. The relevant chromosomal re- gion responsible for the growth advantage of the 15-trisomic cell has not been defined. In order to map this region, we have induced thymic lymphomas by chemical carcinogens (DMBA or MNU) in mice with 2 different constitutional translocations, T(7; 15)OH homozygotes and [T(7; I5)PH x T(5; 15)4Ad] F, hybrids. Twenty-two tumors developed in 90 carcinogen-treated mice. Among the 14 cytogenetically ana- lyzed thymic lymphomas, 4 were diploid and 5 were aneu- ploid, with no chromosome- IS-associated chahges. Five lymphomas showed partial duplication of chromosome 15. Four of them have duplicated the segment distal to the C/DI breakpoint of TPH, while the 5th carried an interstitial dupli- cation of the D2 sub-band of the T(7;IS) translocation chro- mosome. These findings suggest that the duplication of the D 2/3 region, known to contaln the c-myc and the pvt-l genes (Banerjee et a/., 1985). rather than other regions of chromo- some 15, contributes to the development andlor progression of murine T-cell leukemias.

Trisomy of chromosome 15 is the most commpn and often the only chromosomal aberration in murine T-cell lymphomas (Dofuku et al., 1975; Chang et al., 1977; Wiener et al., 1978b,c, 1980). It has been found in spontaneous, virally and chemically induced lymphomas, with frequencies ranging from 40 to 90%, depending on the inducing agent (Dofuku et al . , 1975; Wiener et al., 1978a,b,c, 1980; Herbst et ql., 1981; Herbst and Gropp, 1982; Chan et al., 1979, 1981; Carbonell et al . , 1982, 1987).

The notion that chromosome 15 trisomy can contribute to the development and/or progression of murine T-cell leuke- mias, and is not merely a secondary epiphenomenon, was based on the finding that centromerically fused Robertsonian chromosomes involving number 15(Rb 1;15, Rb 4;15 or Rb 6;15) duplicated in their entirety in virally and chemically induced T-cell lymphomas arising in the constitutional Rb- translocation-carrying mice. Translocations that involved other chromosomes than No. 15 did not duplicate (Spira et al., 1979, 1983; Wiener et al., 1982). Chromosome 15 trisomic T-cell lymphomas induced in reciprocal (14; 15)-translocation-~arry- ing CBAT6T6 mice have duplicated the segment distal to the T6 breakpoint (band B 1/2), indicating that the relevant gene was in the distal part of the chromosome (Wiener et al., 1978b, 1979).

Chromosomes 15, derived from different mouse strains, differ in their propensity to duplicate during T-cell leukemo- genesis in Fl hybrids. The AKR-derived chromosome 15 was found to duplicate preferentially in each F1 combination where the high leukemic AKR strain was one of the parents (Wiener et al., 1979, 1980, 1982; Wirschubsky et al., 1984~). The chromosomes of different strains could be arranged as a con- sistent “hierarchical series”, based on similar preferential du- plications. This can be exemplified by the regular duplication of the APR-derived chromosome 15 in trisomic T-cell leuke-

depending on the genetic origin of chromosome 15. This was an autonomous property of chromosome 15, rather than of the genetic background (Wiener et al . , 1980, 1982).

The present study was performed in an attempt to map the relevant region of chromosome 15 that favors leukemia devel- opment and/or progression after duplication. This was ap- proached by inducing thymic lymphomas in T(7;15)9H and [T(7;15)9H x T(5;15)4 Ad] F1 mice. These constitutional translocations have arisen by more distal breaks on chromo- some 15 than the previously studied T6 translocation. Our findings identify the 15D 213 sub-band region, known to con- tain the c-myc andpvt-1 genes, as the relevant region.

MATERIAL AND METHODS

Mice Two non-inbred translocation-carrying stocks, T(7; 15)9H

and T(5;15)4Ad, were used. T9H breeders with a reciprocal translocation between chromosomes 7 and 15 were obtained from Dr. M. Lyon (MRC Radiobiology Unit, Harwell, UK). Heterozygous males, carrying a reciprocal translocation be- tween chromosomes 5 and 15, were obtained from Dr. I.-D. Adler (Adler and Neuhauser-Klaus, 1984). This translocation was built into the AKR 6; 15 background by repeated back- crossing. It eould readily be identified by conventional Giemsa staining in the [AKR 6;15 X T(5;15)] F1 hybrid offspring.

[T(5;15) X T9H] F1 hybrids were produced by crossing T(7;15)9H homozygotes with [AKR 6;15 X T(5;15)] F! hy- brids. They were karyotyped and mice carrying a single rcpt(7; 15) and a single rcpt(5;15) chromosome were selected for study (Fig. 1).

Lymphoma induction DMBA (7,12-dimethylbenz(a)-antracene) and MNU (methyl-

N-nitroso-N-urea) were administered to different groups of mice. One mg DMBA or 1 mg MNU, dissolved in 0.1 ml polyethylene glycol (wt 400) were given through stomach tube to 6- to 8-week-old mice in 0.1-ml volumes 5 times at weekly intervals (DMBA) or 3 times at monthly intervals (MNU). The majority of the leukemias developed in the thymus, but the spieen and the mesenteric lymph nodes could also appear as the primary site. Chromosome preparation

Metaphase plates were prepared from primary thymic lym- phomas. G-banding was performed as described by Wang and Fedoroff (1971). Chromosomes and bands were identified ac- cording to the criteria of the Committee on Standardized Ge- netic Nomenclature for Mice (1972).

RESULTS Breaboints

chromosome 15 are illustrated in Figure 2. The T( 14; 136, T(7; 15)9H and T(5; 15)4Ad breakpoints on

mias induced in (AKR X CBAT6T6) F1 hybrids, the duplica- tion of the CBAT6T6-derived T( 14; 1 3 6 chromosome in (C57B1 x CBAT6T6) F1 hybrid leukemias, and of the C57B1- derived chromosome 15 in leukemias of (C57B1 x T9H) F1 hybrids. In contrast, genetically identical but cytogenetically different 15-chromosomes duplicated at random. The consis- tency of the “duplication hierarchy” suggested that the rele- vant chromosomal change occurred with a different frequency, Received: August 24, 1987 and in revised form October 20, 1987.

CHROMOSOME 15 TRISOMY IN MURINE T-CELL LYMPHOMA 739

FIGURE 1 - Chromosomal constitution of a [T(7; 15) X T(5; 15)] F1 mouse. Note the presence of both reciprocal translocations.

The T9H breakpoint was early mapped to bands B3 and C on chromosomes 7 and 15, respectively (Dalton et al., 1981). High-resolution banding of the chromosomes involved in this reciprocal translocation confirmed the location of the break- point to the interface of bands C/D1 on chromosome 15. However, slightly different results were obtained for the breakpoint on chromosome 7. This latter was mapped to the interface of bands B5/C as suggested by Dr. E.P. Evans (personal communication).

The T(5;15)4Ad breakpoints were also determined on spreads with a high-resolution banding pattern. They were mapped to 5B and 15 D2 sub-bands, respectively. Lymphoma development

Table I summarizes the results. In contrast to our earlier study where no tumors developed in 44 DMBA-treated T9H mice (Wiener et al., 1982) 4 thymic lymphomas developed in the 21 DMBA-treated T9H mice (19%). Eight of 22 MNU- treated T9H mice developed lymphomas (36%). Both agents were equally effective in F1 hybrids (25% and 20% lympho- mas, respectively). Cytogenetic analysis

The results of the G-banding analyses are summarized in Tables 11 and 111. In Table 111 the tumors were regrouped on the basis of their chromosome constitution and the main chro- mosomal aberration. Four groups could be distinguished. The first group contained 4 tumors, T9H-9, T(5;15)9H-1, 8 and 9. They had a near-diploid chromosome number with minor non- distinctive numerical variations and no structural aberrations.

The 3 tumors in the second group, T9H-1, 4 and 6, con- tained various structural aberrations that did not involve chro- mosome 15. They showed some similarities. T9H-1 lacks one chromosome 16. In T9H-4, one chromosome 16 is either missing or has undergone a reciprocal exchange with chro- mosome 12. T9H-1 also contains a Robertsonian translocation chromosome, generated by centromeric fusion between chro- mosome 6 and an unidentified marker. A similar marker was present in lymphoma T9H-6 as an independent acrocentric element.

Lymphomas of the third group, T9H-8 and T(5;15)9H-5, were trisomic for other chromosomes than 15. T9H-8 was

trisomic for chromosome 14. This anomaly has previously been found in a minority of T- and B-cell lymphomas (Wiener et al., 1981) and was recently identified as a frequent aberra- tion in chemically induced thymic lymphomas (Carbonell et al., 1982, 1987). T9H-8 also contained an extra copy of chromosome 5. T(5; 15)9H-5 was trisomic for chromosome 17. Chromosome 17 trisomy is the second most common nu- merical change in murine T-cell lymphomas, after 15-trisomy (Wiener et al., 1978~).

The fourth group contained 5 lymphomas. Four of them, T9H-10, T(5;15)9H-2, 4 and 7, were trisomic for a given segment of chromosome 15, whereas the fifth, T9H-3, carried an interstitial duplication of the 15 D2 sub-band in one (7;15) chromosome (Fig. 3) leading to the exclusive trisomy of this particular chromosome region.

The 4 chromosome 15-trisomic tumors were subjected to more detailed analysis. The T9H-10 lymphoma contained 2 major groups of metaphase plates. One was diploid without structural aberrations, while the other was hyperdiploid (up to 78 chromosomes). The ratio between chromosomes (7; 15) and (15;7) was 4:2 in 3 hyperdiploid plates. In the total material of 10 analyzed plates, the proportion was 2.9:2.2 (Table 11) indi- cating near trisomy of the (7;15) chromosome.

A 2 l A

15

FIGURE 2 - Schematic illustration of the breakpoints on chromo- some 15 (see text).

740 SILVA E T AL.

TABLE 11 - MEAN CHROMOSOME NUMBER AND THE MAIN CHROMOSOMAL CHANGES IN T9H AND T(5;15)9H LYMPHOMAS

Total number Chromosomes of cells

I 2 3 4 5 6 7 8 9 10 I I 12 13 14 15 16 17 18 19 X Y Tumor

T9H- 1 5 2.0 2.0 2.0 2.0 2.0 1.0 - 2.0 2.0 2.0 2.0 2.0 2.0 2.0 - 1.0 2.0 2.0 2.0 1.0 1.0 T9H-3 12 2.0 1.8 1.9 1.8 1.8 1.9 - 1.9 1.7 1.8 2.0 1.9 1.7 2.0 - 1.9 1.9 2.0 1.9 2.0 - T9H-4 8 2.0 2.0 2.0 1.8 2.1 2.0 - 2.0 2.0 2.0 2.0 1.0 2.0 1.8 - 1.3 2.0 2.0 2.0 1.0 1.0 T9H-6 7 2.0 2.0 2.0 2.0 2.1 2.2 - 2.0 2.0 2.0 2.1 2.0 1.8 1.8 - 1.8 1.8 1.8 2.0 1.7 - T9H-8 15 2.0 2.0 2.0 2.0 3.0 2.0 - 2.0 2.0 2.0 2.0 2.0 2.0 2.9 - 2.0 2.0 2.0 2.0 1.9 - T9H-9 10 2.0 2.0 2.0 2.0 2.0 2.0 - 2.0 2.1 2.0 2.0 1.9 1.9 2.0 - 2.0 2.0 1.9 2.0 2.0 - T9H-10 10 3.3 3.0 2.8 3.1 3.0 3.2 - 2.9 2.9 2.8 3.3 2.9 2.7 2.9 - 2.7 2.9 2.7 3.3 2.5 - T(5; 15)9H- 1 I 1 1.9 2.0 1.8 1.8 1.0 1.9 1.0 1.9 2.0 2.0 1.8 2.0 2.0 1.9 - 2.0 2.1 1.8 2.0 0.9 0.1 T(5; 15)9H-2 9 2.0 2.0 2.0 2.0 1.0 2.0 1.0 2.0 2.0 2.0 2.0 1.7 2.0 2.0 - 2.0 1.0 2.0 2.0 2.0 - T(5; 15)9H-4 6 1.8 2.0 2.0 1.8 1.0 2.0 1.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 - 2.0 2.0 2.0 1.8 2.0 - T(5;15)9H-5 10 2.0 2.2 1.9 2.0 1.0 1.9 1.0 2.1 2.1 2.1 2.2 2.0 2.0 1.9 - 2.0 2.8 2.4 2.0 1.9 - T(5;15)9H-7 11 2.3 2.1 2.1 2.0 1.2 1.9 1.0 2.0 2.0 2.1 2.2 2.0 2.0 2.1 - 1.9 2.0 2.2 2.0 1.8 - T(5;15)9H-8 10 2.0 2.0 2.0 2.0 1.0 2.1 1.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 - 1.9 2.0 2.0 2.0 1.0 0.9 T(5 : 1 S9H-9 10 2.0 1.9 2.0 2.0 1.0 2.0 1.0 2.0 2.0 2.0 1.9 2.0 2.0 2.0 - 2.0 2.0 1.9 2.0 1.0 1.0

TABLE I - LYMPHOMA INCIDENCE IN T(7;15)9H AND [T(7;15)9H X T(5;15)4Ad]F1 HYBIUDS

Strain

T(7;15)9H MNU 2218 (36) 6 T(7; 15)9H DMBA 2114 (19) 1 T(7;15)9H2 DMBA 44/0 (0) - [T(5;15)4Ad X T9HIFl MNU 35/7 (20) 5 [T(5;15)4Ad X T9H]F, DMBA 12/3 (25) 2 ‘Treated mice/lymphoma development.-’Wiener ef al., 1982.

In T(5;15)9H-2 and 4, trisomy 15 was generated by dupli- cation of the distal segment of chromosome 15. It is probable that the (7; 15) chromosome had originally duplicated, but the centromeric segment of chromosome 7 was lost from one of the copies and the distal chromosome 15 segment was translo- cated onto chromosome 17 in T(5;15)9H-2 (Fig. 4) and to chromosome 12 in T(5; 15)9H-4 (Fig. 5). Two metaphase plates of T(5; 15)9H-2 contained an extra segment of chromosome 15 on both chromosomes 17 and 12.

In T(5; 15)9H-7 the T(5; 15) chromosome has duplicated, leading to the trisomy of the bands 15 D 2/3, E and F (Fig. 6 ) .

Thus, 4 of the 5 tumors in the 15-trisomic group have duplicated the segment distal to 15 C/D1. The interstitial duplication of the 15 D2 sub-band in the fifth tumor (T9H-3) identifies D2 as the relevant region.

DISCUSSION

In a previous study (Wiener et al., 1982) we failed to obtain any lymphomas in 44-DMBA-treated T9H mice. The same treatment induced 25 % thymic lymphomas in other genotypes. In F1 hybrid mice between T9H and any of the partner strains C57B1, ASW, CBA or AKR, lymphomas arose in frequencies ranging between 25% (T9H x C57B1)F1 and 45% (T9H X AKR) F1 hybrids, respectively. Twenty of 32 cytogenetically analvzed FI tumors were trisomic for chromosome 15. The 15

FIGURE 3 - G-banded karyotype of T9H-3. Note the interstitial du- plication of the 15 D2 sub-band in one of the rcpt(7; 15) homologues (arrow).

region) to contribute to the DMBA-initiated leukemogenic process. In our present study, MNU was a more efficient inducer of thymic lymphomas than DMBA. All tumors with chromosomal aberrations were in the MNU-treated group (Ta- ble 111), in contrast to our previous study on DMBA-treated C57B1 mice where 100% of the cytogenetically analyzed tu- mors were 15-trisomic (Wiener et al., 1978~).

chrdmosome of the partner strain had duplicated in each case, while the T9H-derived (7; 15) and (15;7) chromosomes re- mained single. This was quite remarkable, since the “hierar- chical position” of the partner strain chromosome 15 ranged from high (in AKR) to low (in C57B1). We have speculated that the constitutional (7; 15) translocation may have impaired the ability of the T9H-derived chromosome 15 (or its relevant

The main purpose of the present study was to narrow down the critical region on chromosome 15 that favors the develop- ment and/or the progression of thymic lymphomas when in the trisomic state. Previously we had shown that this region is distal to the T6 marker breakpoint, generated by a reciprocal (14;15) translocation. This breakpoint is very close to centro- mere, however, in band 15 B1. It was therefore necessary to

CHROMOSOME 15 TRISOMY IN MURINE T-CELL LYMPHOMA 741

TABLE I1 - MEAN CHROMOSOME NUMBER AND THE MAIN CHROMOSOMAL CHANGES IN T9H AND T(5;15)9H LYMPHOMAS (continued)

rcpt rcpt Modal chromosome rcpt rcpt (7;15) (15;7) (5:15) (15;s) (7;dup 15 D2) t(17'ter 15) t(l2;ter 15) (12:16) M 1 Rb(6;Ml) number

- - 2.0 2.0 1 .o 2.0 2.0 2.0 1.7 1.8 2.0 2.0 2.0 2.0 2.9 2.2 1 .o I .o 1 .o 1.0 1 .o 1 .o 1 .o 1.0 1 .o 1 .o 1 .o 1 .o 1 .o 1.2 1 .o 1 .o 1 .o 1.1 1.7 1.0

- - - - - - - - - - - -

- 0.2 1 .o -

1.0 - -

39.0 39.0 40.1 39.8 41.8 39.8 58.0 39.0 40.0 40.6 41.7 41 .S 39.8 39.7

- - - - - - 1 .o 0.9 1 .o 1 .o 1 .o 1 .o 1 .o 1 .o - - - - - -

FIGURE 5 - G-banded karyotype of T(5; 15)9H-4. The telomeric part of the T(7;15)9H parent derived rcpt(7;15) chromosome is translo- cated onto chromosome 12.

FIGURE 4 - G-banded kWoqPe ofT(5;15)9H-2. The telomeric Part of the rcpt(7;15) chromosome IS translocated onto chromosome 17.

perform similar studies on translocations with more distal breakpoints. We have used the constitutional (7; 15) and (5; 15) translocations where the breakpoint is at 15 C/Dl, and at 15 D2, respectively. Our analysis of 4 chromosome 15-trisomic tumors has led to the conclusion that the critical region is on the telomeric side of band 15 D2. This still left the entire telomeric segment, consisting of bands 15 D3, E and F, among the potentially eligible regions. Fortunately, the T9H-3 tumor contained only an interstitial duplication of the 15 D2 band. The restricted trisomy of this region suggests that this sub- band includes the critically important gene@). It is known to contain the c-myc andpvt-1 sequences (Banerjee et al., 1985).

Activation of c-myc by juxtaposition to IgH sequences and of pvt-1 by juxtaposition to the kappa gene is believed to play

an essential role in generating the typical and the variant translocation-carrying forms of mouse plasmacytoma, respec- tively (Klein, 1981; Klein and Klein, 1985; Cory, 1986). The same sequences may be activated in murine T-cell lymphomas by retroviral insertion. The clustering, orientation and struc- ture of the inserted retroviral sequences is consistent with a functional role (Steffen, 1984; Corcoran et al., 1984; Li et al., 1984; Selten et al., 1984; O'Donnel et al., 1985). This is also suggested by the exclusive duplication of the retrovirally rear- ranged, but not the normal myc-carrying 15 chromosome, in all 6 15 trisomic lymphomas studied that had retrovirally rearranged myc-carrying DNA fragments (Wirschubsky et al., 1984b, 1986). Retroviral activation of c-myc and 15 trisomy may be functionally related at least in the tumors where they

742 SILVA ET AL.

TABLE Ill - LATENCY, LYMPHOID TISSUE INVOLVEMENT AND CYTOGENETIC PATTERN OF T(7;15)9H AND T(S;15)4Ad x T(7; 15)9H F, LYMPHOMAS

Tumor Inducing Latency Tissues involved' Chromosome constitution Chromosome agent (days) LN MLN SPL THY Mode Range aberrations

T9H-9 T(5;15)9H-l T(5;15)9H-8 T(5; 15)9H-9 T9H-1 T9H-4 T9H-6 T9H-8 T(5: 15)9H-5 T9H-3' T9H- 10 T(5;15)9H-2

T(5:15)9H-7 T(5;15)9H-4

\ I ,

DMBA MNU DMBA DMBA MNU MNU MNU MNU MNU MNU MNU MNU MNU MNU

152 228 90

156 102 156 127 164 163 171 254 I20 155 189

++ - - ++ ++ ++ + ++ +++ ++ +++ +++

-

-

++ ++++ ++++ ++++ +++ ++++ ++++ ++++ + ++ ++++ ++++ + + +++

39.8 39.0 39.8 39.7 39.0 40.1 39.8 41.8 41.7 39.0 58.0 40.0 40.6 41.5

37-41 39-41 39-41 38-40 39-40 40-41 39-41 41-44 40-50

40-78 37-40

40 40-4 1 40-46

- Rb(6;Ml), - 16 t(12;16), 16q- M I Ts 5, Ts 14 Ts 17 rcpt(7;dup 15 D2) Amplif. of t(7;15) chr. t(l7;ter 15) t(l2;ter 15) Dup t(5;15)

'LN = Lymph node: + = enlarged; ++ = greatly enlarged; - = normal. MLN = Mesenteric lymph node: + = enlarged; ++ = greatly enlarged; - = normal. SPL = Spleen wt: + = 100-300 mg; + + = 600-900 mg; + + + = <900 mg. THY = Thymus wt: + = 50-100 mg; + + = 100-300 mg; + + + = 300-600 mg; ++++ = <600mg.

11 =P' lCplfl6.SI

12

18 d I X

16 17 18

FIGURE 6 - G-banded karyotype of T(5; 15)9H-7. Note the duplica- tion of the rcpt(5;15) chromosome.

coincide. A similar conclusion has been drawn from 2 earlier studies on somatic hybrids, derived from the fusion of 15- trisomic T-cell lymphomas that carried a retrovirally rear- ranged c-myc gene, with normal fibroblasts (Spira et al., 1981; Uno et al., 1987). Weakly tumorigenic hybrids contained rear- ranged and germ-line myc-carrying chromosomes in the same ratios as in the original hybrids, whereas strongly tumorigenic segregants andlor derived in vivo tumors showed an amplifi- cation of the rearranged (R) and often also a decrease in the number of the germ-line (G) myc-carrying chromosomes, with a corresponding change in the R:G ratio (from 2-3:2 to 5- 6:2-1). These findings are consistent with the possibility that an increased dosage of the activated myc gene, or some closely associated sequence, favors leukemic development. Our pres- ent findings provide the first direct support to this conclusion, by showing the selective duplication of the 15 D2 sub-band.

ACKNOWLEDGEMENTS

This investigation was supported by a PHS grant from the National Cancer Institute, DHHS, and by the Swedish Cancer Society. S.S. is the recipient of a fellowship from the Cancer Research Institute and another from Concern Foundation.

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