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[Cancer Research 63, 4895-4898, August 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Measurement of Genomic Instability in Preleukemic P190BCR/ABL Transgenic Mice Using Inter-Simple Sequence Repeat Polymerase Chain Reaction1

Julia M. Brain2, Nathaniel Goodyer and Pierre Laneuville3

Departments of Medicine and the Divisions of Hematology and Molecular Oncology Group, McGill University, Montreal, Quebec, Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BCR/ABL associated leukemias are characterized by a high degree of chromosomal and genomic instability. The genomic instability is usually associated with disease progression, as in chronic myelogenous leukemia or a poor prognosis as observed in hallmark Philadelphia chromosome-positive acute lymphoblastic leukemia. It is unclear whether the phenotype of genomic instability is a primary consequence of Bcr/Abl expression or if it is secondarily acquired in the multistep process of tumor evolution. To address this issue, we measured the frequency of insertions and deletions in P190BCR/ABL transgenic mice. These mice ubiquitously express Bcr/Abl for an average of 3 months before developing B-cell type lymphoma/leukemia. Genome scanning for insertions and deletions in samples of DNA extracted from kidney and spleen tissues taken from preleukemic animals was performed using the inter-simple sequence repeat PCR. We observed an increased frequency of insertions and deletions in the tissues of preleukemic animals, which could be partially reversed with the c-Abl specific inhibitor STI571. These results suggest that the expression of Bcr/Abl can directly induce a mutator phenotype that antedates overt neoplastic transformation, and that STI571 appears to be capable of reversing this effect.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BCR/ABL associated leukemias in humans are characterized by the presence of the translocation t(9;22)(q34;q11) and the cytogenetic Ph4 (1) . This rearrangement inserts the c-ABL gene, which encodes a protein tyrosine kinase, from chromosome 9 into the break cluster region (BCR) gene on chromosome 22 to create a fusion BCR/ABL gene. Depending on the breakpoint position within the BCR gene on chromosome 22, Bcr/Abl fusion proteins with constitutively activated protein tyrosine kinase activity of 190, 210, or 230 kDa are characteristically expressed in acute lymphoblastic leukemia, CML, and chronic neutrophilic leukemia, respectively (2) . The expression of Bcr/Abl is associated with a high degree of chromosomal and genomic instability. This effect is best observed in the acquisition of secondary cytogenetic abnormalities and gene mutations associated with the progression of chronic phase CML to CML blast crisis (3) . The mechanism of this observed genomic instability is unknown.

In addition to the expression of Bcr/Abl, the reciprocal translocation t(9 ,22) results in the variable expression of Abl/Bcr from the derivative chromosome and loss of one normal allele of the BCR and c-ABL genes (4) . The natural history of CML patients expressing Abl/Bcr does not appear to differ from that observed in patients who do not express Abl/Bcr, suggesting that its expression is unlikely to contribute to genomic instability (4) . The loss of one normal BCR allele is also unlikely to play a role. Normal Bcr is ubiquitously expressed and negatively regulates oxidative burst in B cells and neutrophils. Whereas homozygous BCR knock-out mice are susceptible to endotoxin mediated shock, heterozygous BCR knock-out mice appear normal (5) . The c-ABL proto-oncogene, however, has been implicated in cell-cycle regulation and response to DNA damage after genotoxic stress. Over-expression of c-Abl induces G1 cell-cycle arrest, whereas deficiency of c-Abl confers a susceptibility to enhanced cellular transformation by dominant oncogenes (2 , 6 , 7) . Thus, both the expression of the dominant oncogene BCR/ABL and the concurrent loss of an allele of the tumor suppressor gene c-ABL could potentially directly initiate genomic instability in Ph-positive leukemias. Conversely, genomic instability may result from secondary genetic or epigenetic events (8 , 9) .

In previous studies, we have shown that retroviral transduction and expression of Bcr/Abl encoding P210 into 32D cl3(G) cells results in inhibition of apoptosis altered cell cycle regulation and induce rapid numerical and structural chromosomal abnormalities (10 , 11) . More recently, we measured the basal point-mutation rate in heterozygote P190BCR/ABL/LIZ transgenic mice. The chromosomally integrated {lambda} bacteriophage shuttle vector (LIZ) contains a bacterial lacI gene as a target for mutation and {alpha}lacZ as a reporter gene (i.e., Big Blue mutation detection system; Stratagene). The line of P190BCR/ABL mice used express Bcr/Abl ubiquitously before developing B-cell lymphoma/leukemia with a latency of ~100 days (12) . In this preleukemic period, when there is no sign of cellular transformation, we observed a 2–3-fold steady-state increase in the frequency of point mutations in P190BCR/ABL/LIZ mice compared with control (13) . Although these studies demonstrated that the expression of Bcr/Abl can directly induce a mutator phenotype antedating overt leukemic transformation, the experimental system used restricted characterization of this mutator phenotype to the study of point mutations only.

In the present study, we address the question of genomic instability by using inter-SSR PCR, in combination with primers which consist of a set of eight CA repeats (Fig. 1ACitation ; see Ref. 14 ). These CA repeats appear in all species tested and are the most frequent repeats in the human genome, with an estimated copy number of 50,000–100,000 per haploid genome (15) . Using Inter-SSR PCR, we compared the P190BCR/ABL preleukemic mice and P190BCR/ABL leukemic mice with control mice (BL6/CBA) and found an increased number of altered bands (insertions and deletions) in the preleukemic and leukemic mice compared with control. We also show that the frequency of altered bands can be decreased using the c-Abl specific kinase inhibitor STI571. These results confirm that the expression of Bcr/Abl alone can confer genomic instability before the onset of leukemia, and also extends the list of DNA repair defects characterizing the Bcr/Abl induced mutator phenotype to include double stranded DNA breaks.



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  		Fig. 1. A, inter-SSR PCR is a highly effective method for detecting differences within (CA)n repeats. The two primers consist of CA repeats of different lengths and amplify the region of DNA within these repeats. B, gel-banding patterns observed for normal mouse spleen (mouse number = 72) and kidney (mouse number = 95). The 18 bands shown were used to score for the presence of insertions and deletions in test samples. C, typical RG primer-banding pattern from kidney samples of control and a P190 transgenic mouse. A deletion is present in the P190BCR/ABL mouse sample.

 

   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Mice/DNA Isolation.
Transgenic P190BCR/ABL (line 623) were kindly donated by Dr. John Groffen (Children’s Hospital, Los Angeles, CA). The derivation and phenotype of this mouse line has been described extensively elsewhere (16) . These mice are characterized by the ubiquitous expression of Bcr/Abl that is driven by a truncated mouse metallothionein promoter, which precedes the onset of a B-cell lymphoma/leukemia with a latency of ~100 days after birth. BL6/CBA mice were used as controls (Jackson Lab, Bar Harbor, ME). The mice were genotyped by using tail DNA and Southern blot analysis. Preleukemic mice were sacrificed and the kidneys and spleen removed for whole genomic DNA isolation using the Wizard DNA Isolation Kit (Promega, Madison, WI). Tumor tissue isolated from mice that had undergone leukemic transformation was processed similarly.

STI571 Kinase Inhibitor.
The c-Abl-specific kinase inhibitor STI571 (formally known as CGP57148B) was from Novartis Pharmaceuticals (Basel, Switzerland). A stock solution of STI571 was prepared by dissolving 35.7 mg of STI571 in 1 ml of 100% DMSO. The injection solution was prepared by making a final concentration of 10% DMSO and STI571 of the initial stock in sterile PBS. Mice were weighed and received i.p. injections of 50 mg/kg STI571 daily for 10 consecutive days. This dose of STI571 has been shown previously to completely inhibit Bcr/Abl in a mouse tumorigenic assay (17) .

PCR.
PCR amplification was carried out using 1 µM RG primer [(CA)8RG]; 50 ng of genomic DNA; 0.3 units Taq polymerase (Life Technologies, Inc., Rockville, MD) and 1 µCi of 32P{alpha}dCTP (250 µCi; Amersham, Arlington Heights, IL) in a 20-µL total mixture of PCR buffer [10 mM Tris-HCL (pH 9.0); 2% formamide; 50 mM KCL; 0.2 mM deoxynucleoside triphosphates; 1.5 mM MgCL2; 0.01% gelatin; 0.01% Triton X-100]. The primer RG consists of eight CA repeats anchored by two nucleotides, in which G is guanine and R is a 50:50 mixture of the purines adenine (A) and guanine (G; Fig. 1ACitation ).

Amplification was performed using a Perkin-Elmer Cycler (Cetus), with an initial denaturation for 3 min at 94°C, followed by 30 PCR cycles at 94°C for 30 s, at 52°C for 45 s, and at 72°C for 2 min. A final extension at 72°C was performed for 7 min.

Gel Analysis.
The PCR product was loaded on an 8% nondenaturing polyacrylamide gel, run at constant 1500 V, dried and exposed to film (Biomax, Kodak, Amersham, Arlington Heights, IL) at room temperature for 2 days. The gels were analyzed using one normal control as a standard for each tissue (Fig. 1B)Citation . Using this control, an average of 20 bands were counted and compared with each sample run to count insertions and deletions (Fig. 1C)Citation . A blind reading of the samples was performed to avoid bias in reading the results. One control was used and compared with other controls to look for variance within each control. Repeat analyses of all samples were performed (average of five PCR reactions per tissue sample) to minimize the effect of experimental variability. Data analysis was completed and the statistical calculations were carried out using Microsoft Excel and SigmaPlot.

Light-Cycler RT-PCR.
Total RNA was extracted from kidney and spleen samples using TRIzol (Life Technologies, Inc., Rockville, MD). RNA concentration was determined by spectrophotometric measurements at A260 and a ratio of A260/A280. The integrity of the RNA was tested by running 5 mg of total RNA on a formaldehyde gel. Quantification and RT-PCR was performed per the manufacturer’s instructions (Roche Molecular Biochemicals).

Sequencing.
Band products were gel extracted from a 1% agarose gel and clones were prepared in SK+pbluescript (Stratagene, La Jolla, CA) using the T7 primer-forward (New England BioLabs, Beverly, MA) and the T3 primer-reverse (New England BioLabs, Beverly, MA). Sequencing was performed at the Sheldon Biotechnology Centre (Montreal, Quebec, Canada).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Quantification of Bcr/Abl in the Various Tissue Samples.
To determine the relative differences between expression of Bcr/Abl in various tissues, a quantitative RT-PCR was performed using a Light-Cycler Quantitation Kit. When comparing the preleukemic spleen and leukemic spleen, there was an increased expression of Bcr/abl in the leukemic spleen (Table 1)Citation . Also noteworthy was the fact that when comparing the preleukemic spleen to the preleukemic kidney, there was a much higher expression of Bcr/Abl in the kidney than in the spleen.


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  		Table 1 Relative levels of Bcr/Abl as detected by LightCycler analysis

RNA samples were used to quantitate levels of Bcr/Abl in each tissue.

 
Demonstration of Genomic Instability in Preleukemic P190BCR/ABL Transgenic Mice Spleen.
To determine whether the P190BCR/ABL transgenic mice accumulated mutations in lymphocytes before developing B-lymphocyte leukemia/lymphoma, we examined DNA isolated from the spleen of preleukemic P190BCR/ABL transgenic mice for the presence of insertions and deletions using Inter-SSR PCR. As shown in Fig. 2ACitation , the results were compared with those observed in normal control spleen and B-cell tumors and spleens from P190BCR/ABL mice that had undergone leukemic transformation. Whereas no insertions or deletions were observed in control mice spleen DNA, spleens from P190BCR/ABL preleukemic mice contained an average of three such events (P = 0.0006). The observed rate of insertions and deletions in P190BCR/ABL preleukemic mice was approximately half that observed in tumors (P = 0.003). These results suggest that Bcr/Abl expression can directly induce genomic instability characterized by DNA insertions and deletions before leukemic transformation. To examine whether this instability could be decreased using a c-Abl specific inhibitor, we gave mice injections of the inhibitor STI571. We observed an increased frequency of insertions and deletions in control mice injected with STI571 (P = 0.00), but no difference in the preleukemic P190BCR/ABL mice with or without treatment with STI571 (P = 0.46). This was an unexpected result that raises the possibility that inhibition of normal c-Abl may be mutagenic in normal spleen lymphocytes (i.e., STI571 is equally effective at inhibiting normal c-Abl and Bcr/Abl proteins). This observation could be in keeping with the tumor-suppressor role some investigators have assigned to c-Abl (7) .



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  		Fig. 2. A, observed insertions and deletions from samples amplified with the RG primer. Samples include control spleen (Control; n = 3) from normal mouse strain (BL6/CBA) and preleukemic (PL; n = 5) or leukemic spleens (L Spl; n = 3) from P190BCR/ABL transgenic mice with (Inj; n = 3) or without injection (n = 3) with the kinase inhibitor, STI571. Also included are tumor samples from lymphatic tissue (Tumor; n = 9). B, observed insertions and deletions from samples amplified with the RG primer. Samples include control kidney (Control; n = 3) from normal mouse strain (BL6/CBA) and preleukemic (PL; n = 5) kidney (Kid) from P190BCR/ABL transgenic mice with (n = 3) or without injection (Inj; n = 3) with the kinase inhibitor STI571.

 
Demonstration of Genomic Instability in Preleukemic P190BCR/ABL Transgenic Mice Kidney.
To determine whether the P190BCR/ABL transgenic mice similarly accumulated mutations in nonlymphoid, nontransforming Bcr/Abl expressing tissues, we examined DNA isolated from preleukemic P190BCR/ABL transgenic mouse kidneys using inter-SSR PCR (Fig. 2B)Citation . Previous studies in our laboratory (unpublished data) have shown high expression of Bcr/Abl in the kidney by Western blot analysis and have demonstrated that this was associated with an increased frequency of point mutations, even though oncogenic transformation was restricted to B lymphocytes. We observed an increased frequency of altered bands in the preleukemic P190BCR/ABL mice compared with control (P = 0.007). A basal rate of insertions and deletions could be detected in normal control kidney DNA that was not observed in normal spleen tissue.

In contrast to what we observed in normal spleen tissue, the basal mutation rate was not increased in the kidneys of mice treated with STI571 (P = 0.897). This difference might be explained by the masking of a mutagenic STI571 effect by the basal mutation rate observed in control kidney, by insensitivity of kidney tissue to the loss of normal c-Abl function, or by some other mechanism. The mutation frequency in preleukemic kidney tissue was higher than that observed in spleen, and it was possible to show that this could be partially reduced with STI571 treatment (P = 0.045).

Taken together, these results confirm the ability of Bcr/Abl to directly increase the frequency of insertions and deletions. Although our results indicate the potential beneficial effect of STI571 to decrease this effect by inhibiting Bcr/Abl, the observed increase in mutation frequency in normal spleen tissue of mice treated with STI571 raises some concern that inhibition of normal c-Abl may be mutagenic in susceptible cells.

One way to confirm these results would be to address the mutation frequencies and the effects of STI571 in other hematopoietic tissues. We have attempted this on bone marrow samples of these transgenic mice in our laboratory, and our preliminary studies indicate that several mouse samples would need to be combined for these studies, and, in this case, we cannot rule out the possibility of differences between individual mice. Therefore, we believe the spleen was the best tissue to observe hematopoietic changes.

Specificity of Inter-SSR PCR Assay.
To determine whether this assay was efficient at amplification of CA repeats, several bands were cloned and sequenced to address their specificity. In each of the sequence samples cloned, the CA-repeat primer was noted, as well as identical sequences for bands which were run on the same gels (Fig. 3)Citation . These experiments indicated that this PCR assay was very specific for the amplification of CA repeats and that the bands being compared are of identical sequences.



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  		Fig. 3. Sequence analysis confirms specificity of experiment. Bands were extracted from a 1% agarose gel from a normal (BL6/CBA) and a P190BCR/ABL preleukemic mouse. The bands were extracted from side-by-side experiments, and the bands removed appeared at the same molecular mass. Both samples clearly show the CA repeats as well as identical, opposing sequences.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Bcr/Abl expression in human leukemias, animal models of leukemia, and cell transformation systems has been associated with genomic instability. CML remains one of the classic examples demonstrating the role of genomic instability in tumor progression, as evidenced by the increased frequency of cytogenetic, genetic, and epigenetic alterations that accompany transition from the chronic phase of the disease to its invariably fatal blast cell crisis (8) . Genomic instability is a prominent hallmark of neoplastic evolution, which generally facilitates the acquisition of successive mutations that lead to increased deregulation of cell growth and differentiation. In some instances, the initiating mutation is directly responsible for inducing genomic instability, as exemplified by many of the inherited cancer prone disorders including ataxia telangiectasia, Bloom’s syndrome, xeroderma pigmentosum, and DNA mismatch-repair defects (18, 19, 20) . More commonly, the mutation(s) implicated in promoting genomic instability is secondarily acquired after tumor initiation (i.e., loss of p53 tumor suppressor function).

Whether Bcr/Abl is a direct inducer of genomic instability or is secondarily acquired has been the subject of a recurring debate. In the present study, we addressed this issue by taking advantage of the P190BCR/ABL line 623 transgenic mice that express Bcr/Abl before developing leukemia with an average latency of 100 days after birth. The level of Bcr/Abl expression in these mice is much lower than that commonly observed in vector-transformed Bcr/Abl expressing cell lines, thus minimizing the risk of artifacts resulting from overexpression. We have closely examined these animals and have not been able to demonstrate defects in apoptosis, cell-cycle regulation, or response to genotoxic stress at these levels of Bcr/Abl in preleukemic mice (21) . However, the level of Bcr/abl expression is sufficient to induce leukemia. The only abnormality that we have observed is a subtle abnormality of bone marrow B-lymphocyte development (22) . Using this animal model system in a previous study, we have shown a 2–3-fold increase in the point-mutation frequency in the spleen and kidney (13) . In the present study, using inter-SSR PCR, we were able to show that increased frequency of insertions and deletions occur in both spleen and kidney tissues before neoplastic transformation, providing strong evidence that Bcr/Abl can directly induce genomic instability. The mutation frequency in kidney was greater than that observed in spleen tissue and was consistent with the higher expression of Bcr/Abl observed (Table 1)Citation .

In contrast to BCR/ABL positive ALL, most patients with chronic phase CML present with leukemic cells that still retain most of the functions of their normal hematopoietic cell counterparts before recognizable secondary cytogenetic and genetic changes occur. Thus, the inhibition of Bcr/Abl would be expected not only to suppress the clonal expansion of myeloid cell growth, but also to reverse the mutator phenotype conferred by Bcr/Abl as observed experimentally, reduce the rate of secondary genetic events, and either prolong the chronic phase, or block progression to CML blast crisis. Treatment with the c-Abl specific kinase inhibitor STI571, which is currently undergoing clinic trials, has proven highly effective in inhibiting myeloid cell growth in CML. It is still too early to know whether this beneficial effect can be sustained over the long term or whether secondary neoplastic transformation can be prevented. In the present study, we attempted to reverse the mutator phenotype observed in preleukemic P190BCR/ABL transgenic mice using injections of STI571. We were able to show a partial reversal of the mutation frequency in preleukemic kidney, but not in spleen cells, perhaps attributable, in part, to the higher basal mutation frequency observed in the former. This is the result expected for inhibition of the dominant Bcr/Abl oncoprotein. However, we also observed an increased frequency of insertions and deletions in normal spleen cells treated with STI571 compared with untreated controls. Given that STI571 is equally effective at inhibiting c-Abl as it is Bcr/Abl, the increase in mutations observed in normal spleen cells associated with STI571 treatment raises the possibility that inhibition of c-Abl, a putative tumor suppressor gene, may have deleterious effects in some tissues. This observation and the risk of inducing secondary cancers from the pharmacological inhibition of c-Abl in normal tissues deserves further investigation.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Sponsored by grants from the Medical Research Council of Canada. Back

2 Present address: The Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115. Back

3 To whom requests for reprints should be addressed, at Division of Hematology, Royal Victoria Hospital, 687 Pine Avenue West, Room C6.82, Montreal, Quebec H3A 1A1, Canada. Phone: (514) 843-1558; Fax: (514) 843-1418; E-mail: laneuvillep{at}muhchem.mcgill.ca Back

4 The abbreviations used are: Ph, hallmark Philadelphia chromosome; CML, chronic myelogenous leukemia; SSR, simple sequence repeat; RT-PCR, reverse-transcription PCR. Back

Received 2/13/01. Revised 5/27/03. Accepted 6/11/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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