We previously described a preleukemic state induced by Moloney murine leukemia virus (Mo-MuLV) characterized by hematopoietic hyperplasia in the spleen.
JOURNAL OF VIROLOGY, Aug. 1991,
p.
Vol. 65, No. 83
x.I-
4442-4448
r
{
IL XT
n
0022-538X/91/084442-07$02.00/0 Copyright ©) 1991, American Society for Microbiology
Bone Marrow Depletion by 89Sr Complements a Preleukemic Defect in a Long Terminal Repeat Variant of Moloney Murine Leukemia Virus QI-XIANG LI AND HUNG FAN* and Biochemistry and Cancer Research Institute, University of California, Biology Department of Molecular Irvine, California 92717 Received 15 October 1990/Accepted 9 May 1991
We previously described a preleukemic state induced by Moloney murine leukemia virus (Mo-MuLV) characterized by hematopoietic hyperplasia in the spleen. Further experiments suggested that splenic hyperplasia results from inhibitory effects in the bone marrow, leading to compensatory extramedullary hematopoiesis. An enhancer variant of Mo-MuLV, Mo+PyF101 Mo-MuLV, fails to induce preleukemic hyperplasia and has greatly reduced leukemogenicity, indicating the importance of this state to efficient leukemogenesis. An alternative method for induction of preleukemic hyperplasia was sought. Treatment of mice with 89Sr causes specific ablation of bone marrow hematopoiesis and compensatory extramedullary hematopoiesis in spleen and nodes. NIH Swiss mice were inoculated neonatally with Mo+PyF101 Mo-MuLV and treated with 89Sr at 6 weeks of age. Approximately 85% developed lymphoid leukemia with a time course resembling that caused by wild-type Mo-MuLV. In contrast, very few animals treated with Mo+PyF101 MoMuLV or 89Sr alone developed disease. In approximately one-third of cases, the Mo+PyF101 Mo-MuLV proviruses were found at common sites for wild-type Mo-MuLV-induced tumors (c-myc, pvt-1, and pim-1), indicating that this virus is capable of performing insertional activation in T-lymphoid cells. These results support the proposal that splenic hyperplasia results from inhibitory effects in the bone marrow. They also indicate that Mo+PyF101 Mo-MuLV is blocked in early and not late events in leukemogenesis.
undergoes subsequent infection, this can result in proviral insertion near a proto-oncogene such as c-myc or pim-J (6, 19). This results in transcriptional activation of the protooncogene(s) and outgrowth of the final tumor. The importance of preleukemic hematopoietic hyperplasia was highlighted by the fact that Mo+PyF101 Mo-MuLV was unable to induce preleukemic splenic hyperplasia (7), although it could efficiently establish infection in thymocytes (8). Thus, the leukemogenic defect in Mo+PyF101 MoMuLV appeared to be in a preleukemic event. On the other hand, Mo+PyF101 Mo-MuLV seemed theoretically capable of activating proto-oncogenes in target thymocytes, since it established the same high-level infection in thymocytes at preleukemic times as did wild-type Mo-MuLV, although with a slight delay (8). Recent investigations of Mo-MuLV-induced splenic hyperplasia suggested an indirect and growth-inhibitory mechanism. Splenic hyperplasia could not be attributed to direct infection of hematopoietic progenitors, since progenitors from nonhyperplastic spleens of Mo+PyF101 Mo-MuLVinfected mice showed the same high level of infection as those from hyperplastic spleens of Mo-MuLV-infected mice (3). This suggested that infection of other cells was necessary for hematopoietic hyperplasia. In addition, somewhat surprisingly, in vitro long-term bone marrow cultures from preleukemic Mo-MuLV-inoculated mice showed a quantitative defect in hematopoiesis in comparison with control or Mo+PyF101 Mo-MuLV-infected animals (18). The longterm bone marrow culture defect was the result of combined infection by Mo-MuLV and an MCF recombinant. These results raised the possibility that Mo-MuLV induces splenic hyperplasia by inhibiting bone marrow hematopoiesis. Splenic (extramedullary) hematopoiesis could be a physiological response to reduced bone marrow hematopoiesis.
Retroviruses that lack oncogenes induce neoplasms with long latency. The latency is at least partly due to the multistep nature of leukemogenesis by these viruses. Some of these steps include insertional activation of cellular protooncogenes by retroviral long terminal repeats (LTRs) (6, 16) and (for murine leukemia viruses) generation of env gene recombinants (mink cell focus-forming virus [MCF]) (15). In studies of Moloney murine leukemia virus (Mo-MuLV) leukemogenesis, we and others also documented an early hematopoietic hyperplasia occurring in the spleen (7, 27). A key in our experiments was an LTR variant of Mo-MuLV, Mo+PyF101 Mo-MuLV, in which transcriptional enhancer sequences from the F101 strain of polyomavirus were inserted into the Mo-MuLV LTR by molecular cloning (21). Mo+PyF101 Mo-MuLV has altered transcriptional control sequences, but virus particles are physically identical to wild-type Mo-MuLV virions, since the alteration is in a region which does not encode viral proteins. Mo+PyF101 Mo-MuLV is poorly leukemogenic, and it also does not induce preleukemic hyperplasia (7, 9). In previous studies on Mo-MuLV leukemogenesis (using Mo+PyF101 Mo-MuLV as a tool), we proposed a twoinfection model for disease induction (7). In the first infection, Mo-MuLV directly or indirectly induces early splenic hyperplasia involving multiple hematopoietic lineages (7, 27), even though Mo-MuLV ultimately induces exclusively T-lymphoid leukemia under standard conditions. The splenic hyperplasia is evident as splenomegaly and as elevated numbers of myeloid and erythroid hematopoietic progenitors in agar colony assays. In the second infection, if a hyperplastic lymphoid progenitor migrates to the thymnus and *
Corresponding author. 4442
89Sr INDUCTION OF PRELEUKEMIA
VOL. 65, 1991
In this report, we provide evidence supporting this. Mo+PyF101 Mo-MuLV has greatly increased leukemogenicity in mice treated with 89Sr, a nonviral means of inducing splenic hyperplasia.
MATERIALS AND METHODS
Viruses, cells, and animals. Wild-type Mo-MuLV and Mo+PyF101 Mo-MuLV used in these experiments were described before (9, 18). Viral stocks were clarified tissue culture supernatants from productively infected NIH 3T3 cells. Neonatal NIH Swiss mice were inoculated subcutaneously with these viruses (approximately 105 XC PFU per animal) within 48 h of birth (7). 89Sr treatment of mice. Virus-inoculated or uninoculated mice were injected intraperitoneally with 10 pLCi of 89Sr chloride (Amersham, Inc.) in 0.2 ml of phosphate-buffered saline at weekly intervals beginning 6 weeks after birth. At 10 days after the first injection, two mice were sacrificed and hematopoietic progenitors in bone marrow and spleen were assayed (see below). The rest of the mice were monitored for tumor development. Moribund mice were sacrificed, and tumor tissues were analyzed (see below). Hematopoietic colony assays. Assays for mixed colonyforming cells (CFCmix) in spleen and bone marrow were performed as described previously (7). Briefly, single-cell suspensions from spleen or bone marrow were suspended in soft agar in growth medium containing WEHI-3B cell supernatant as a source of interleukin-3. Colonies (greater than 50 cells) were scored microscopically after 7 days of incubation.
Analysis of tumor DNAs. High-molecular-weight DNA was extracted from tumor tissues as described previously (14). After digestion with various restriction enzymes, DNAs were analyzed by gel electrophoresis and Southern blot hybridization (25) with different 32P-labeled probes. The probes were nick-translated restriction fragments or endlabeled synthetic oligonucleotides. The following probes were used: for Mo-MuLV DNA, the PstI fragment from 563 to 739 bp from the pMLVlA molecular clone (21); for the T-cell-receptor beta (TCR-P) gene, the 600-bp EcoRI constant-region fragment from plasmid 86T5 (17); for immunoglobulin mu heavy chain (IgH), the 700-bp XbaI-EcoRI fragment containing sequences immediately 3' to the J region from plasmid p2-1 (30); for polyomavirus sequences, a synthetic 30-bp oligonucleotide from the core of the inserted sequences; for pvt-1, the 2.2-kb XbaI fragment from the probe b plasmid clone (5); for pim-1, the 1-kb BamHI fragment from the probe A clone (6); and for c-myc, the 2-kb XhoI fragment from the pM c-myc 54 cDNA clone (26). RESULTS
89Sr-induced splenic hyperplasia in mice. One implication of the two-infection model for Mo-MuLV leukemogenesis would be that the Mo+PyF101 Mo-MuLV variant should be highly leukemogenic if preleukemic splenic hyperplasia could be induced by other means. We therefore tested whether nonviral induction of extramedullary hematopoiesis (and splenic hyperplasia) could yield a preleukemic state for Mo+PyF101 Mo-MuLV. 89Sr is a low-energy radioisotope that homes to the bone and selectively ablates bone marrow hematopoiesis (1). Treatment of mice with 89Sr has been shown to induce elevated splenic hematopoiesis for multiple lineages as well (1, 23a). As shown in Table 1, adult mice inoculated with 10 ,uCi of 89Sr showed 5- to 10-fold decreases
4443
TABLE 1. Hematopoietic progenitors in "9Sr-treated mice"
CFCmix Treatment
None Mo+PyF101 Mo-MuLV
89Sr Mo+PyF101 Mo-MuLV plus 89Sr
Bone marrow (per 105 cells)
Spleen (per 8 x 105 cells)
42 37 8 3
9 8 68 46
" NIH Swiss mice were injected subcutaneously with 105 XC PFU of Mo+PyF101 Mo-MuLV at birth and/or injected intraperitoneally with 10 ,Ci of 89Sr at approximately 6 weeks of age. Ten days after the '9Sr injection (or at an equivalent time), animals were sacrificed and bone marrow or spleen cells were assayed for myeloid progenitors (CFCmix) as described previously (3). The CFCmix assays were agar colony assays in medium containing WEHI-3B cell supernatant as a source of interleukin-3. Mo+PyF101 MoMuLV infection had little effect on the number or distribution of CFCmi,x while treatment with '9Sr decreased bone marrow and increased splenic hematopoiesis.
in bone marrow myeloid progenitors (CFCmix) and corresponding increases in splenic CFCmix within 10 days. The 5to 10-fold increases resembled the elevation of splenic CFUmix in preleukemic mice inoculated with Mo-MuLV (7). Others have shown that 89Sr-induced changes in hematopoiesis are transitory (1). 89Sr complements Mo+PyF101 Mo-MuLV for leukemogenesis. Neonatal NIH Swiss mice were inoculated subcutaneously with an Mo+PyF101 Mo-MuLV stock at birth. Beginning at 6 weeks of age, four weekly injections of 10 ,uCi of 89Sr were administered to induce sustained splenic hyperplasia. As shown in Fig. 1, animals showed tumor development beginning within 1 month after completion of the 89Sr treatment, with 15 of 17 eventually dying. Mean time to disease was 17 weeks, very similar to the 15-week mean time for wild-type Mo-MuLV-induced leukemia in previous studies (11). In contrast, only 3 of 15 mice inoculated with Mo+PyF101 Mo-MuLV alone developed tumors during the study. Thus, 89Sr treatment complemented the preleukemic defect of Mo+PyF101 Mo-MuLV. Mice treated with 89Sr alone also developed tumors, but with a significantly greater latency (more than 28 weeks for the first tumor), consistent with previous reports (31). All tumors were lymphoblastic lymphoma by histopathology. Characterization of tumors in 89Sr-treated Mo+PyF101 Mo-MuLV-inoculated mice. Tumors from the animals described in Fig. 1 were characterized for virological status and tissue type by Southern blot hybridization, as summarized in Table 2. To test for the presence of Mo+PyF101 Mo-MuLV DNA in tumors, we screened samples for the presence of a diagnostic 1.9-kb PvuII fragment recognized by an MoMuLV gag probe (Fig. 2A). Uninfected NIH 3T3 cell DNA showed no hybridizing bands in the 1.5- to 2.0-kb region, while the corresponding band from wild-type Mo-MuLVinfected cells (lane 43-D) migrated at 1.7 kb. Almost all the 89Sr plus Mo+PyF101 Mo-MuLV tumors showed the diagnostic 1.9-kb PvuII fragment, which comigrated with the fragment from Mo+PyF101 Mo-MuLV-infected NIH 3T3 cells (lane 25-3). In addition, some tumors showed additional fragments suggestive of deletions in the LTR (e.g., tumor 284-1); LTR rearrangements in tumors induced by other enhancer variants of Mo-MuLV have been previously observed (14). An alternative explanation for some of the apparent LTR variants could have been recombination with endogenous MuLV proviruses. In any event, when 89Sr plus Mo+PyF101 Mo-MuLV-induced tumors were examined
4444
J. VIROL.
LI AND FAN 100
90
n.17
p
70
60
% Tumor Mortality 50s
n13
40
n-15
30 20 10
0
0
2
4
6
8
10
12
16
14
18
20
22
24
26
28
30
32
Weeks Injecffon of 89Sr
FIG. 1. 89Sr complements Mo+PyF101 Mo-MuLV for leukemogenesis. Newborn NIH Swiss mice were inoculated subcutaneously with 105 XC PFU of Mo-MuLV. Beginning at 6 weeks of age, four weekly injections of 10 ,uCi of 89Sr (aqueous solutions of 89Sr chloride [Amersham, Inc.] diluted into phosphate-buffered saline) were administered. For comparison, animals also were infected with Mo+PyF101 Mo-MuLV alone or injected with 89Sr alone. Two litters of mice were used for each experimental group, and the total number of animals is indicated (n). Moribund animals were sacrificed and subjected to necropsy, histopathology, and molecular analyses. The time course of animals dying with tumors is shown. W1, 89Sr plus Mo+PyF101 Mo-MuLV; O, Mo+PyF101 Mo-MuLV; A, 89Sr.
with a polyomavirus-specific DNA probe, they showed evidence of Mo+PyF101 Mo-MuLV infection as well (e.g., see Fig. 5). These results indicated that the tumors contained Mo+PyF101 Mo-MuLV proviral DNA, consistent with viral induction of the tumors. Tumor DNAs were also analyzed for the presence of MCF recombinant viruses by Southern blot analysis (4). These represent env gene recombinants between the infecting MuLV and endogenous MuLV proviruses. We recently showed that Mo+PyF101 Mo-MuLV does not generate MCF recombinants in vivo (4). As summarized in Table 2, none of the tumors showed evidence of MCF recombinants. The histopathology of the tumors indicated lymphoblastic lymphoma. To determine whether they were T-lymphoid or B-lymphoid or null cell derived, we tested tumor DNAs for rearrangements of the TCR-P or -lgH genes. The generally accepted criterion is that T-lymphoid tumors will show TCR gene rearrangements, with or without lgH gene rearrangements. B-lymphoid tumors will show germ line TCR gene configurations but will have IgH with or without immunoglobulin light-chain gene rearrangements. Therefore, all tu-
first tested for TCR-,B gene rearrangements, as illustrated in Fig. 3. Those tumors showing germ line TCR-P configurations were then also tested for lgH gene rearrangements, as illustrated in Fig. 4. Most of the tumors induced by Sr89 plus Mo+PyF101 Mo-MuLV were T lymphoid, similar to wild-type Mo-MuLV-induced lymphomas, while approximately one-third were B lymphoid. The rare tumors resulting from Mo+PyF101 Mo-MuLV inoculation alone were T lymphoid. On the other hand, tumors ultimately developing from 89Sr inoculation alone (including additional ones not summarized in Table 2) were exclusively B lymphoid. The B lymphomas induced by 89Sr plus Mo+PyF101 Mo-MuLV might reflect a shift in the enhancer specificity of the Mo+PyF101 Mo-MuLV LTR relative to the wild-type MoMuLV LTR; others have shown that the tissue specificities of leukemias resulting from MuLVs are determined by the enhancer specificities of their LTRs (10). Alternatively, 89Sr treatment might establish a preleukemic condition that favors development of B-lymphoid tumors. Proviral insertions near proto-oncogenes. If Mo+PyF101 Mo-MuLV was performing typical late steps in leukemogenmors were
TABLE 2. Tumors in 89Sr plus Mo+PyF101 Mo-MuLV-treated micea No. with tumors Treatmt
Mo+PyF101 Mo-MuLV 89Sr plus Mo+PyF101 Mo-MuLV
3/15 15/17
Tumor type T lymphoid B
3/3 9/14
Virus
lymphoid 0/3 5/14
Mo+PyF101 Mo-MuLV 3/3
15/15
MCF recombinant
0/3 0/15
a High-molecular-weight DNAs from the tumors induced by 89Sr plus Mo+PyF101 Mo-MuLV or Mo+PyF101 Mo-MuLV alone were analyzed by Southern blot hybridization. The presence of Mo+PyF101 Mo-MuLV provirus in the tumors was assayed by digestion with SmaI or XbaI followed by hybridization with an Mo-MuLV env region probe. In this analysis, Mo+PyF101 Mo-MuLV proviruses yield a diagnostic 2.2-kb internal fragment, as described previously (11). The presence of MCF recombinants was assayed by digestion with XbaI plus BamHI and hybridization with an MCF env probe, which would yield a diagnostic 2.2-kb fragment. We recently found that Mo+PyF101 Mo-MuLV does not generate MCF recombinants in vivo (4). Digestion of the tumor DNAs and hybridization with TCR-13 or immunoglobulin heavy- or light-chain probes was performed as described previously (13). Tumors with TCR-, gene rearrangements were classified as T lymphoid, while those with germ line TCR-p genes but with rearrangements in immunoglobulin heavy- and/or light-chain genes were classified as B lymphoid
(13).
89Sr INDUCTION OF PRELEUKEMIA
VOL. 65, 1991 A
Xba
Xba I
PstI
t
4445
=-
Pvu 11
XbalI
- (---Drt--4=
Pvu II
Py sequences
Pvu 11
-
Pvu 11
-do
Pvu 1I
23 w.t. Mo >
t
w
+~~6.7kb 4 2.3 kb 420 kb
FIG. 2. Provirus in tumors-gag and 5' LTR analysis. (A) Maps of wild-type (w.t.) and Mo+PyF101 Mo-MuLVs (Mo+Py) in DNA form. Restriction sites for PvuII are shown. The location of the hybridization probe is also indicated (dark solid box). Wild-type Mo-MuLV gives a diagnostic 1.7-kb fragment, while Mo+PyF101 Mo-MuLV gives a 1.9-kb fragment. (B) Tumor or control DNAs were digested with PvuII and analyzed by agarose gel electrophoresis and Southern blot hybridization with the 5' gag probe. NIH 3T3, uninfected cell control; 43-D, NIH 3T3 cells infected with wild-type Mo-MuLV; 25-3, NIH 3T3 cells infected with Mo+PyF1O1 Mo-MuLV; 265-2 to 285-5, tumor DNAs from 89Sr plus Mo+PyF101 Mo-MuLV-inoculated mice; 275-1, tumor DNA from a mouse inoculated with Mo+PyF101 Mo-MuLV alone. The gag probe also hybridizes with endogenous MuLV-related proviral DNA present (predominantly in restriction fragments of 5 to 15 kb), present in all lanes. The diagnostic fragments are indicated by the arrows. The apparent higher mobility of the 1.9-kb fragment for tumor 285-4 is an electrophoresis artifact-see the mobility of the faint band at 2.4 kb.
esis in 89Sr-treated mice, then the tumors might be expected to show insertional activation of proto-oncogenes. Therefore, a collection of 89Sr plus Mo+PyF101 Mo-MuLV tumors was screened by Southern blot analysis for proviral insertion near the three common insertion sites for MoMuLV-induced lymphomas in mice: c-myc, pvt-J, and pim-1 (6, 13, 23). Representative analysis of tumors showing insertions near pim-J, c-myc, and pvt-J are shown in Fig. 5. The tumor DNAs showed novel-sized proto-oncogene restriction fragments in comparison with nontumor DNA, presumably resulting from insertion of an Mo+PyF101 Mo-MuLV provirus nearby. As expected, hybridization of the same blot with an oligonucleotide probe specific for the polyomavirus enhancer showed hybridization at the same positions as for the novel proto-oncogene-hybridizing fragments. This was
FIG. 3. TCR-P rearrangements in the tumors. Tumor DNAs were digested with HpaI and analyzed by Southern blot hybridization with a TCR-] constant-region fragment probe (2). Unrearranged germ line bands at both 11.6 and 6.1 kb are indicated. In NIH Swiss mice, there is also a restriction polymorphism, resulting in 11.6- and 12.2-kb HpaI fragments (2). NIH 3T3, NIH 3T3 cell DNA, with germ line fragments; 267-3, tumor from an animal treated with 89Sr alone; 275-2 and 275-3, tumors from mice inoculated with Mo+PyF101 Mo-MuLV alone; 285-2 and 284-7, tumors from 89Sr plus Mo+PyF1l1 Mo-MuLV-inoculated mice. All lanes were from the same Southern blot, but the photographic exposures were adjusted to account for differential DNA loading. Tumors 275-2, 275-3, and 285-2 were classified as T lymphoid.
consistent with the rearranged fragments containing a Mo+PyF101 Mo-MuLV provirus. As shown in Table 3, a significant fraction (approximately one-third) of 89Sr plus Mo+PyF101 Mo-MuLV tumors showed insertion near c-myc, pim-J, or pvt-J. This was a lower limit on the percentage of tumors showing insertional activation, since other insertion sites (e.g., those for MoMuLV-induced tumors in rats [28, 29]) were not tested. Also, it was possible that the B lymphomas had insertional
z
-
a. 4 5-.D ,
_U-4earraMn3ed am
_4
.-
-,-S
amp.
so
-4
2.3 K
FIG. 4. IgH gene rearrangements in tumors. Tumor DNAs that did not show TCR-P gene rearrangements were digested with EcoRI and analyzed by Southern blot hybridization with an 1gH probe. The unrearranged 6.4-kb germ line band is indicated. NIH 3T3, control NIH 3T3 cells; 267-1 and 267-2, tumors from animals treated with 89Sr alone; 284-4 to 255-1, tumors from 89Sr plus Mo+PyF101 Mo-MuLV-inoculated animals. These were all B lymphoid by this criterion.
J. VIROL.
LI AND FAN
4446
i
'
:.
__:
.1
1
4
r',
.
W
.1
5
,^*t_i
