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Expression of the Wilms’ Tumor Suppressor Gene, WT1, Reduces the Tumorigenicity of the Leukemic Cell Line M1 in C.B-17 scid/scid Mice¹

Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Queensland 4029, Australia [S. I. S., M. D., A. W. B., C. L. L.], and Departments of Medicine [A. W. B.] and Pathology [C. L. L.], University of Queensland, St. Lucia, Queensland 4072, Australia

	ABSTRACT

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The Wilms’ tumor suppressor gene, WT1, encodes a transcription factor of the Cys₂-His₂ zinc finger type. Loss of WT1 gene function has been implicated in the development of malignancies including Wilms’ tumor and acute leukemias. We have shown previously that ectopic expression of WT1 +KTS isoforms in murine M1 leukemic cells spontaneously induces monocytic differentiation without the requirement for external differentiation-inducing stimuli. To determine whether these observed effects in vitro corresponded to a reduction in tumorigenicity in vivo, parental M1, control M1.Neo, and M1.WT1 +KTS cells were transplanted into C.B-17 scid/scid mice, and the growth and metastatic behavior of the cell lines were monitored for a period of 20 weeks. Mice inoculated either s.c. on the flank or directly into the peritoneal cavity, with M1 cells stably expressing WT1 +KTS isoforms exhibited a marked decrease in tumor formation compared with control groups. Moreover, tumors arising in mice after the injection of M1.WT1 +KTS cells exhibited a loss in ectopic WT1 protein expression. Confirmation that the tumors arose from M1.WT1 +KTS cells was achieved by the amplification of the introduced transgene from tumor samples and indicates that the tumorigenicity of leukemic M1 cells in these animals correlates with a loss in WT1 expression. This investigation is the first to demonstrate the tumor-suppressive effects of WT1 expression in a leukemic cell line, further advancing the notion that WT1 acts as a differentiation-promoting gene during hematopoiesis and that loss of functional WT1 expression may contribute to leukemogenesis in vivo.

	Introduction

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Implicated in the onset of WT,³ a pediatric nephroblastoma, the WT1 gene is a critical regulator of cellular proliferation and differentiation within the genitourinary system (reviewed in Ref. 1 ). The WT1 gene encodes a Cys₂-His₂ ZF transcription factor, and two alternative splice sites within the gene produce the four major WT1 polypeptides: ±17 amino acids encoded by the presence or absence of exon 5 and inserted NH₂-terminal of the four contiguous ZF DNA-binding domains; and ±3 amino acids (KTS) encoded by the presence or absence of 9 bp at the end of exon 9 and inserted between the third and fourth ZF DNA-binding domains (2) . In the developing kidney, WT1 is expressed in the early metanephric stem cells as they condense around the ureteric bud and undergo differentiation into renal epithelial structures, with expression eventually restricted to the podocyte layer of mature glomeruli (3) . WT1 transcripts can also be found in the primitive genital ridge tissue that gives rise to mature gonadal structures (3) . Accordingly, homozygous inactivation of WT1 in mice results in the failure of both kidney and gonadal formation (4) . The necessity for functional WT1 expression during normal genitourinary development is best reflected at a clinical level, with gross deletions and intragenic mutations detectable at the WT1 11p13 locus in at least 5% of WT cases and >95% of patients with Denys-Drash syndrome (WT in association with genital abnormalities and nephropathy; reviewed in Ref. 5 ). The expression of WT1 in both normal and neoplastic hematopoietic cells (6 , 7) has led to speculation that this gene may play a similar role in regulating cellular proliferation and differentiation during blood cell development. Mutations within the WT1 gene have been detected in 12% of acute leukemia patient samples primarily of biphenotypic (lymphoid/myeloid) or early myeloid origin (8 , 9) . Furthermore, expression and/or mutation of WT1 in acute leukemias may indicate the presence of minimal residual disease and predict a poor prognosis for these patients (8, 9, 10) .

The observations from the clinical situation, combined with its function as a transcriptional repressor, have resulted in the classification of WT1 as a tumor suppressor gene, whereby loss of functional WT1 gene expression is sufficient for initiation of tumorigenesis. The paradigm of the tumor-suppressive effect of WT1 within genitourinary development has been well defined. Indeed, ectopic expression of normal WT1 transcripts in various malignant kidney cell lines has primarily resulted in a marked suppression of the tumorigenic phenotype of the cells upon s.c. transplant into animal recipients (11, 12, 13) . To date, however, no studies have been conducted to ascertain whether WT1 has the potential to perform a similar tumor-suppressive function in leukemic cell lines. The murine myeloblastic leukemia cell line M1 forms myelosarcomas upon syngeneic transplantation into SL mice (14 , 15) . Induction of cellular differentiation in M1 cells by exposure to differentiation inducers leads to a marked suppression of leukemogenicity (16 , 17) . Using these cells as an in vitro model for macrophage differentiation, we have shown previously that parental M1 cells, which lack endogenous expression of WT1 in an undifferentiated state, express high levels of WT1 after induction of macrophage differentiation by LIF (18) . Furthermore, it was demonstrated that the ectopic expression of WT1 +KTS isoforms in M1 cells induced monocytic differentiation without the requirement for external differentiation-inducing stimuli (18) . In this investigation, we now demonstrate that M1.WT1 +KTS cells exhibit a marked reduction in tumorigenicity compared with parental and control M1 cells when allogeneically transplanted into immunodeficient SCID mice. This study is the first to demonstrate that WT1 can abrogate the tumorigenic potential of a leukemic cell line and therefore suggests that this gene may act, as it does in the developing kidney, as a classical tumor suppressor gene during blood cell development, with the functional loss of WT1 contributing to the onset of leukemogenesis in vivo.

	Materials and Methods

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Cells and Cell Culture.
The myeloblastic leukemia cell line M1 was established from a spontaneous myeloid leukemia that arose in the SL strain of mice (14) . The generation, maintenance, and in vitro characterization of M1 cells stably transfected with expression vectors containing WT1 +KTS cDNAs or a control neomycin-resistance cDNA have been described in detail elsewhere (18) .

Clonogenicity Assay.
Clonogenic potential was assessed by colony formation in soft agar. After washing, 200 cells in 1 ml of Iscove’s modified Dulbecco’s medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS (HyClone Laboratories, Inc., Logan, UT) and 0.3% Bacto-Agar (Difco Laboratories, Detroit, MI) were plated in duplicate into 35-mm Petri dishes. Recombinant murine LIF (a gift from AMRAD Biotech, Melbourne, Australia) was added to experimental cultures at a concentration of 1 ng/ml. Plates were kept at 37°C in a humidified atmosphere with 5% CO₂, and after 7 days, colonies were scored as clones containing >50 cells, whereas abortive clones containing <50 cells were scored as clusters.

Tumorigenicity Assay.
Animal studies were performed with appropriate institutional ethics committee approval and in accordance with National Health and Medical Research Council guidelines. Specific pathogen-free SCID mice were obtained from the Animal Resources Center (Perth, Australia) and housed in air-filtered cages under sterile conditions. M1, M1.Neo, M1.WT1.1(+/+), and M1.WT1.2(-/+) cell lines for in vivo transplantation were harvested, washed, and resuspended in PBS. For s.c. injections, adult female SCID mice were inoculated with 1 x 10⁶ cells in 100 µl of PBS on the right dorsal flank. For i.p. injections, age-matched 6-week-old female SCID mice were inoculated with 1 x 10⁶ cells in 100 µl of PBS. Control mice not receiving injections received PBS alone. For each site tested, six mice/group received injections, and the experiments were performed twice at separate intervals. The results were then combined to give a total number of 12 mice/group for each site. Tumor formation and tumor-related mortality were monitored twice weekly for a period of 20 weeks, after which time any mice showing an absence of tumor incidence were considered to be tumor free.

Histology.
For histological examination, mice were autopsied upon humane sacrifice, and any tumorous material was removed. Tissues were fixed in PBS-buffered 10% formalin and embedded in paraffin, and 5-µm sections were stained with Mayer’s H&E using standard techniques.

Protein Extraction and Western Blotting.
Analysis of WT1 expression in tumors derived from injection of M1.WT1 +KTS cells was facilitated by the extraction of protein from paraffin-embedded tumor tissue (19) . Briefly, 50 µM sections were deparaffinized using standard techniques, followed by homogenization in radioimmunoprecipitation assay buffer containing 2% SDS [2% SDS, 1% sodium deoxycholate, 1% Triton X-100 (Sigma Chemical Co., St. Louis, MO), 150 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4] and the Complete Protease Inhibitor Cocktail (Boehringer Mannheim, Mannheim, Germany). Samples were incubated at 100°C for 20 min, followed by 2 h at 60°C. Protein extraction from cultured cell lines and tumor ascites was performed as described previously (18) . After centrifugation, total protein (25 µg) was then separated on an SDS-10% polyacrylamide gel and subjected to immunoblotting with the WT (C-19) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), also as described previously (18) . Protein recovery was monitored with a cross-specific rabbit anti-actin antibody (Sigma Chemical Co.) using a similar immunoblotting procedure.

Genomic DNA Extraction and PCR.
The detection of the WT1 transgene in tumors derived from injection of M1.WT1 +KTS cells was facilitated by the extraction of genomic DNA from paraffin-embedded tumor tissue (20) . Briefly, 50-µm sections were deparaffinized using standard techniques, followed by incubation in TNE buffer containing 1% SDS (1% SDS, 5 mM EDTA, 150 mM NaCl, and 50 mM Tris-HCl, pH 8.0) and 500 µg/ml proteinase K (Merck, Darmstadt, Germany) for 48 h at 45°C. An additional 250 µg/ml of proteinase K was added to the samples on each successive day of incubation. DNA extraction from cultured cell lines and tumor ascites was performed as described previously (18) . After organic extraction and ethanol precipitation, 500 ng of genomic DNA was used in a PCR incorporating a 5' primer directed to exon 7 of the WT1 transgene 5'-CCCAGGCTGCAATAAGAGATA-3', and a 3' primer directed to the retroviral expression vector, 5'-AGCCTGGACCACTGATATCC-3'. Conditions for WT1 amplification, in addition to amplification of the murine ß-actin gene to monitor DNA integrity were also as described previously (18) . PCR products (5 µl; 10 µl for ß-actin PCR on genomic DNA from paraffin-embedded tumor tissue) were separated on 1% agarose gels in 1x TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.5).

Statistical Analysis.
The significance of differences in the proportion of mice that developed tumors or survived injections between the control and experimental groups was determined using the nonparametric Mann-Whitney U test. The control groups of mice receiving injections, inoculated with either M1 or M1.Neo cells, were combined for the purposes of this analysis. P < 0.05 was considered significant.

	Results

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Expression of the WT1 + KTS Isoforms Leads to a Reduction in Clonogenicity of M1 Cells in Soft Agar.
As a measure of clonogenic potential, the ability of M1 cells stably transfected with the +KTS isoforms of WT1 to form colonies in soft agar was compared with parental M1 cells and control M1.Neo cells. Colony formation was assessed in either the presence or absence of LIF. In the absence of LIF, M1 and M1.Neo cells exhibited overall plating efficiencies of 50 and 49%, respectively (Fig. 1A) . For M1 cells, compact, undifferentiated colonies represented 88% of cell growth in these cultures, and 95% of M1.Neo growth also exhibited this phenotype (Fig. 1) . Despite the observation that M1.WT1.1(+/+) and M1.WT1.2(-/+) cells exhibited a reduction in plating efficiency compared with control cells (34 and 16%, respectively; Fig. 1A ), the proportion of colonies in these cultures, a mixture of compact and diffuse-type colonies (Fig. 1B) , was still significant at 76% for M1.WT1.1(+/+) cells and 63% for M1.WT1.2(-/+) cells. Although the decreased ability of M1.WT1 +KTS cells to grow in soft agar correlates with an increased differentiative phenotype in these cells (Fig. 1B) , it is important to note that sequential replating of differentiated colonies from these cultures resulted in the regeneration of secondary colonies that exhibited both nondifferentiated and differentiated morphologies (data not shown). Furthermore, we had observed previously that the growth of M1.WT1 +KTS cells in liquid culture did not significantly differ from control M1 cells. Unlike M1 cells induced for macrophage differentiation by LIF, these cells do not exhibit clonal exhaustion associated with terminal macrophage differentiation (18) . These results would suggest that although ectopic WT1 +KTS isoform expression promotes the differentiation of M1 cells along the monocyte/macrophage pathway, it still enables the maintenance of viable, self-renewing stem cells within this population, thus facilitating their persistence in culture as a continuously proliferating cell line.

View larger version (71K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. A, colony formation in soft agar by M1 cells is markedly reduced by the expression of WT1 +KTS isoforms, particularly in the presence of LIF. Cells were plated in the presence or absence of LIF, and after 7 days, colonies were scored as clones containing >50 cells, whereas abortive clones containing <50 cells were scored as clusters. , colonies; , clusters. B, photomicrographs showing the morphology of the various M1 cell populations upon soft agar culture in the absence of LIF. Colonies shown are representative of those observed throughout the culture. Unstained. x40.

Expression of WT1 +KTS Isoforms Leads to a Reduction in Tumorigenicity of M1 Cells upon s.c. Injection into C.B-17 scid/scid Mice.
To determine the effect of WT1 +KTS isoform expression on the in vivo growth and metastatic properties of M1 cells, the various M1 cell-derived populations were injected s.c. into SCID mice. Whereas 11 of 12 mice (92%) and 12 of 12 mice (100%) receiving injections with parental M1 and control M1.Neo cells, respectively, formed tumors, the incidence of tumor formation in mice injected with M1.WT1 +KTS cells was greatly reduced (Table 1) . Tumors arose in only 3 of 12 mice (25%) in the group transplanted with M1.WT1.1(+/+) cells, a reduction in tumor formation of up to 75% over control groups. Moreover, in the cohort injected with M1.WT1.2(-/+) cells, tumor formation was completely abolished. No mice in the PBS control group were affected (Table 1) .

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. The survival of SCID mice receiving injections s.c. on the dorsal flank (A) or i.p. (B) with M1 cells is significantly enhanced by the expression of WT1 +KTS isoforms. Mice were injected with the various M1 cell populations, as described in "Materials and Methods," and monitored for tumor-related mortality for a period of 20 weeks. , PBS; , M1; •, M1.Neo; , M1.WT1.1(+/+); , M1.WT1.2(-/+).

View larger version (141K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Photomicrographs showing the histology of tumors resected from SCID mice receiving injections s.c. on the dorsal flank with the various M1 cell populations. M1.Neo and M1.WT1.1(+/+) cells formed myelosarcomas either localized to the site of injection (A and C, respectively) or viscerally disseminated within mesenteric membranes of the peritoneal cavity, leading to the neoplastic infiltration of associated organs such as the duodenum and stomach (B and D, respectively). H&E stain. x100.

However, evaluation of the median latency period for onset of tumorigenesis at this site did not reveal the marked differences observed at the s.c. site between the control and experimental groups (Table 1) , with all four groups giving comparable mean times of tumor onset (49–60 days). The cumulative survival of SCID mice injected i.p. with the M1 cell populations is presented in Fig. 2B and further emphasizes the overall increase in survival of mice inoculated with M1.WT1 +KTS cells.

The presentation of myelosarcomas within the peritoneal cavity after the direct i.p. injection of cells closely resembled that of myelosarcomas that had formed at this location after injection of cells at the s.c. site, with all cell populations preferentially lodging within the mesenteric membrane, often leading to the invasion of adjacent organs such as the kidney and duodenum (data not shown). However, additional peritoneal tumors were also observed within the pancreas and the adipose tissue surrounding the uterine horns.

Loss of Ectopic WT1 Expression in Tumors Arising from the Injection of M1.WT1 +KTS Cells.
Because tumors were still observed upon injection of the transfected population of M1.WT1 +KTS cells, it was of interest to determine whether these tumors were in fact derived from clones of cells that had altered levels of expression of the WT1 transgene compared with the original cell line injected. This was assessed by immunoblotting of protein and PCR amplification of genomic DNA isolated from the injected M1.WT1 +KTS cell lines, an ascitic tumor that arose in a mouse receiving an injection of M1.WT1.1(+/+) cells at the s.c. dorsal site, and solid tumors that arose in mice receiving injections of either M1.WT1.1(+/+) or M1.WT1.2(-/+) cells at the i.p. site. In all tumors examined, ectopic WT1 protein expression was either not detectable (in M1.WT1.1(+/+) tumors from both the s.c. and i.p. injections) or dramatically reduced [in an M1.WT1.2(-/+) tumor from the i.p. injection] in comparison with the level of ectopic WT1 expression (visualized as M_r 54,000/52,000 protein products) in the original cell lines at the time of injection (Fig. 4A) . The level of endogenous actin expression (a M_r 42,000 protein product) is also shown to indicate the efficiency of protein recovery from each sample (Fig. 4A) . To confirm that these tumors did indeed arise from M1.WT1 +KTS cells, PCR amplification of genomic DNA isolated from these tumors was performed to detect the presence of the integrated transgene containing the WT1 cDNAs (visualized as a 540-bp PCR product; Fig. 4B ), thereby indicating that tumors in mice receiving injections of M1.WT1 +KTS cells arose from M1 cell clones that showed inadequate levels of WT1 +KTS isoform expression. Amplification of ß-actin (a 1.2-kb PCR product) is also shown as an indicator of genomic DNA recovery from each sample (Fig. 4B) .

View larger version (59K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Western and PCR analysis of the M1.WT1 +KTS cell lines and tumors subsequently derived from their injection into SCID mice demonstrate a loss of ectopic WT1 expression in the tumor samples, despite the presence of M1.WT1 +KTS cells. Total protein was extracted from cell lines, tumor ascites, or solid tumors, and duplicate blots were hybridized with antibodies to WT1 (A, upper panel) to detect ectopic WT1 expression or actin (A, lower panel) to demonstrate equivalent protein recovery and loading between samples. Genomic DNA isolated from identical samples was subjected to PCR using primers directed toward the integrated WT1 transgene (B, upper panel) to detect the presence of M1.WT1 +KTS cells in the tumors or ß-actin (B, lower panel) to indicate the quality of DNA recovered from the samples.

	Discussion

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Because of the plethora of data implicating WT1 in the etiology of WT, previous studies on the ability of specific WT1 isoforms to suppress cellular tumorigenicity have focused on malignant kidney-derived cell lines (11, 12, 13) , although the ability of WT1 to inhibit tumor growth of ras^H-transformed NIH3T3 cells has also been investigated (23) . This present study is the first to extend this assessment to include a tumor cell line of hematopoietic origin, and a comparison of WT1 isoform tumor-suppression activity in cell lines examined to date is shown in Table 2 . In summary, all WT1 isoforms have exhibited the capacity to suppress tumor formation in at least one cell type; however, the degree of protection afforded by each isoform varies according to the cell line under investigation. Across both renal and hematopoietic cell types, the WT1.2(-/+) isoform appears to be able to uniformly suppress tumor cell growth quite markedly, or even, as in the case of our current study in M1 cells, completely at one site of injection. The WT1.1(+/+) isoform can also function as a tumor suppressor in both cell types tested, although the effect is not as pronounced as with the WT1.2(-/+) isoform. The ability of the WT1.3(+/-) isoform to suppress tumor cell growth in vivo has only been examined in kidney-derived cell lines and is reasonably effective in abrogating tumorigenesis in these cells. The WT1 isoform that lacks both splice inserts, WT1.4(-/-), has demonstrated the ability to circumvent oncogenic transformation of NIH3T3 cells by Harvey ras. However, in adenovirus-transformed kidney cells, this isoform seemingly promotes tumor cell growth in vivo. This result is quite curious, given the growth-suppressive effects that have been observed across a range of cell types forced to express WT1 -KTS isoforms (reviewed in Ref. 24 ). Clarification as to the role of this isoform in either preventing or promoting tumor cell growth can only come through a more extensive analysis in additional cell lines.

In summary, this investigation is the first to demonstrate the tumor-suppressive effects of WT1 expression in a leukemic cell line. As assessed by both in vitro and in vivo assays, ectopic expression of WT1 +KTS isoforms has the potential to revert the leukemic phenotype of myeloblastic M1 cells. This study further advances the notion that, as in the developing kidney, WT1 acts as a tumor suppressor gene during hematopoiesis, whereby loss of functional gene expression may contribute to leukemogenesis in vivo.

	ACKNOWLEDGMENTS

 
We thank Dr. Elizabeth Algar, Dr. Melissa Little, and Prof. David Hume for helpful discussion and Dr. Gu Zhu for assistance with the statistical analysis. The excellent histology work performed by Estelle Schoch and Michael Walsh, and the veterinary pathology advice provided by Dr. John Finnie, are also gratefully acknowledged.

	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 Supported by the Queensland Cancer Fund, the Leukaemia Foundation of Queensland, and the National Health and Medical Research Council of Australia.

² To whom requests for reprints should be addressed, at Queensland Institute of Medical Research, Post Office, Royal Brisbane Hospital, Herston Road, Herston, Queensland 4029, Australia. Phone: 61-7-3362-0321; Fax: 61-7-3362-0105; E-mail: shirleyS{at}qimr.edu.au

³ The abbreviations used are: WT, Wilms’ tumor; ZF, zinc finger; KTS, lysine-threonine-serine; LIF, leukemia inhibitory factor; SCID, C.B-17 scid/scid.

Received 8/16/99. Accepted 1/ 4/00.

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