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[Cancer Research 62, 575-579, January 15, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Loss of the p21Cip1/Waf1 Cyclin Kinase Inhibitor Results in Propagation of Horizontally Transferred DNA1

Anna Bergsmedh, Anna Szeles, Anna-Lena Spetz and Lars Holmgren2

Cancer Center Karolinska Hospital, Karolinska Institutet, S-171 76 Stockholm [A. B., L. H.]; Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 77 Stockholm [A. S.]; and Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, University Hospital, S-141 86 Huddinge [A-L. S.], Sweden


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown previously that phagocytosis of cells dying by apoptosis results in transfer of whole or fragments of chromosomes into the nucleus of the recipient cell. Although DNA transfer was detected in normal cells, stable propagation of the transferred DNA was only observed in cells deficient in p53. Here we show that mouse embryonic fibroblast cells lacking the p21 (Cip1/Waf1) cyclin-kinase inhibitor are able to propagate DNA engulfed by phagocytosis of apoptotic bodies. Feeding mouse embryonic fibroblast p21-/- cells with apoptotic bodies derived from a rat fibrosarcoma resulted in focus formation in vitro and tumor formation in vivo. In contrast, cells lacking the p19 alternative reading frame gene did not show any evidence of transformation. These data indicate that p53, via the activation of p21, blocks normal cells from replicating transferred DNA from engulfed apoptotic bodies. This may be one protection level against the propagation of potentially pathological DNA.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor progression has been described by Foulds as the development of a tumor by a series of permanent irreversible qualitative changes (1) . These perturbations usually involve the deregulation of cell proliferation, up-regulation of telomerase activity, escape from apoptosis, and the induction of angiogenesis (2) . Because of the low mutation rate in normal cells, it has been proposed that specific mutations may result in genomic instability and increased mutability. The most common form of genomic instability is detected at the chromosomal level as the gain or loss of whole chromosomes, chromosomal translocations, or gene amplifications (3) . Several molecular explanations for chromosomal instability have been proposed, e.g., changes in cellular processes involved in the replication and separation of chromosomes during mitosis may result in chromosomal instability. In addition, loss of the p53 tumor suppressor gene in mouse fibroblasts correlates with aneuploidy of cells cultured in vitro (4) .

Analysis of cell proliferation in human tumors indicates that a substantial fraction of cells are continuously eliminated by programmed cell death or apoptosis (5) . Although tumors have a high level of apoptosis, it is usually difficult to distinguish because of the rapid clearance by phagocytes or neighboring cells (6) . It has been generally considered that DNA from dying cells is degraded after apoptosis and, thus, inactivated. However, we have shown previously that genetic information may be transferred from dying to living cells via the uptake of apoptotic bodies (7, 8, 9) . The transfer of drug resistance genes has also been proposed to occur via uptake of apoptotic bodies (10) . These findings indicated that horizontal transfer of DNA from apoptotic bodies could be one explanation to the chromosomal instability observed in cancer cells (7 , 8) . The transfer of DNA is a very efficient process because cocultivation of endothelial cells with apoptotic bodies derived from a human Burkitt’s lymphoma cell line resulted in uptake of apoptotic DNA in >15% of the cells (7) . Recently, we have shown that cocultivation of apoptotic bodies derived from an H-rasV12, human c-myc-transfected rat fibrosarcoma cell line transforms cells deficient in p53 but not wt3 mouse embryonic fibroblasts. Injection of MEF p53-/- cells cocultivated with apoptotic bodies derived from an H-rasV12- and human c-myc-transformed fibrosarcoma cell line in SCID mice resulted in tumor formation (8) . Consistent with our previous data, transfer of apoptotic DNA to wt MEF cells resulted in cell cycle arrest and senescence (7 , 8) . These findings indicate that DNA derived from apoptotic bodies trigger cell cycle arrest or senescence in a p53-dependent pathway.

The p19 ARF and p21 (Cip1/Waf1) genes have been implicated in the activation of p53-dependent senescence (11 , 12) . Overexpression of p19ARF in MEF cells drives the cells into a senescent state. The p19ARF protein binds mdm2, thus preventing mdm2 binding p53 resulting in increased protein stability (13 , 14) . This results in accumulation of p53 and activation of p53 target genes. One of the p53 target genes is p21, which was initially identified in senescent cells and has been implicated in p53-mediated growth arrest of senescent cells. We show here that p21-deficient cells are able to propagate DNA that has been engulfed by phagocytosis. These cells show similar frequencies of focus formation or uptake of the gene encoding hygromycin resistance to that of MEF p53-/- cells. These data indicate that p53 via the activation of p21 normally blocks cells from propagating engulfed apoptotic DNA.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Cell Culture.
REFs and MEFs from wt p53-/-, p21-/-, and p19ARF-/- mice were grown in DMEM (LifeTechnologies, Inc.) with 10% fetal bovine serum (LifeTechnologies, Inc.), glutamine, and penicillin/streptomycin. REFrm cells are REF cells transfected with the H-rasV12, human c-myc (kindly provided by Dr R. N. Eisenman), and a hygromycin resistance gene fused with green fluorescence protein (pEGFP-hygr; ClontechLaboratories, Inc.) using the calcium phosphate transfection method (15) .

Immunofluorescent Stainings.
MEF, MEF p53-/-, MEF p21-/-, and MEF p19ARF-/- cells were plated in a chamber slide (Falcon) and incubated overnight in 37°C, 10% CO2. The cells were irradiated the following day with a dose of 60 Gy. Cells were fixed in 3.7% formaldehyde 3 h after irradiation. p53 induction was analyzed by immunofluorescent staining. Irradiated or nonirradiated cells were incubated with the PAb421 anti-p53, monoclonal antibody (Oncogene Research Products) for 30 min directly after blocking with 10% horse serum. Positive staining was detected with an antimouse FITC-conjugated secondary antibody (DAKO) for 30 min at room temperature. The nuclei were counterstained with Hoechst 33258. The slides were examined with a Leica DMRBE microscope equipped with a cooled CCD camera (Hamamatsu 4800) and filter sets specific for the fluorochromes (DAPI, FITC, and Spectrum Orange).

Gene Transfer Experiments.
For coculture experiments, 2 x 106 MEF or MEF p53-/- cells were trypsinized and transferred to 10-cm Petri dishes. Apoptosis was induced in 10 x 106 REFrm and REF nontransfected cells by irradiation (150 Gy). Irradiated cells were incubated with MEF, MEF p53-/-, MEFp21-/-, or MEF ARF-/- cells at a ratio of 5:1 directly after irradiation. The tissue culture media were changed after 48 h and then changed every 3 days. Some of the cells were grown in the presence of hygromycin (200 µg/ml) to select for the uptake of hygromycin resistance gene. Focus formation was scored as described after 14 days in culture (8) .

PCR Analysis and Immunoblotting.
DNA was isolated with Qiaamp Blood Kit (Qiagen), and PCR analysis was performed with specific primers for human T24 H-ras (5'-ggcaggagaccctgtaggag-3', 3'-gtattcgtccacaaaatggttct-5'), human c-myc (5'-gaggctattctgcccatttg-3', 3'-cagcagctcgaatttcttcc-5'), and hygr (5'-acgtaaacggccacaagttc-3', 3'-aagtcgtgctgcttcatgtg-5'). Conditions for PCR amplification were as follows: H-rasV12: 30 s 95°C, 45 s 61°C, and 2 min 72°C for 30 cycles and human c-myc and hygr: 30 s 95°C, 45 s 58°C, and 2 min 72°C for 30 cycles. For immunoprecipitation and Western blot analysis, 3 x 107 cells were lysed in RIPA buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, and 0.5% aprotinin] and incubated at 4°C for 10 min. The cell suspension was homogenized by repeated aspiration through a 21-gauge needle. The cells were centrifuged for 15 min, 3000 rpm, at 4°C. The supernatant was incubated with antibody for 1 h at 4°C (human H-ras R02120; Transduction Laboratories and c-myc (N-262) sc-764; Santa Cruz Biotechnology, Inc.); 35 µl of ImmunoPure Immobilized Protein G (20398; Pierce) was added to each sample and incubated overnight at +4°C. The precipitates were centrifuged for 5 min, 2500 rpm, and washed twice in RIPA buffer before washes in high salt wash [10 mM Tris-HCl (pH 7.4), 2 M NaCl, 1% NP40, and 0.5% sodium deoxycholate] and low salt wash (1% NP40, 0.5% aprotinin, and 10 mM NaF), once in 1 M MgCl2, once in 1 mM Tris (pH 7.5), and, finally, once in RIPA buffer. Immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis as described previously. Filters were blocked with 5% milk (Bio-Rad nonfat dry milk, 170-6404) for 1 h at room temperature, and the membrane was incubated with the primary antibody [pan-H-rasV12, OP38; Oncogene Research Products and c-myc (9E10); Santa Cruz Biotechnology, Inc.] overnight at 4°C. After repeated washes in 0.2% Tween 20 in PBS, the membrane was incubated with secondary antibody antimouse-horseradish peroxidase (NA 931; Amersham Pharmacia Biotech and Life Sciences, Inc.) for 2 h at room temperature. The membrane was developed using the enhanced chemiluminescence detection system according to protocol of the manufacturer (RPN2209; Amersham Pharmacia Biotech).

Tumorigenicity Assays.
Immunodeficient SCID mice were maintained in a pathogen-free environment. ~4 x 106 MEF, MEF p21-/-, MEF p19ARF-/-, and MEF p53-/- cells cultivated with apoptotic REFrm cells were injected into the dorsal s.c. space of 6–7-week-old SCID mice. Tumor growth was measured with a caliper every 3rd day. The tumor volume was measured by the formula: width2 x length x 0.52 cm3. The animals were killed when the tumors reached a diameter of 2 cm.

FISH Analysis.
The rat genomic DNA was labeled by nick translation with biotinylated-16-dUTP (Boehringer Mannheim) using the BIONICK labeling kit (Life Technologies, Inc.). The human c-myc DNA probe was labeled with Spectrum Orange (Vysis). Interphase nuclei and metaphase chromosomes were analyzed on standard cytogenetic preparations, as described previously (16) . Hybridization was performed in 50% formamide, 2 x SSC, and 10% dextran sulfate at 37°C overnight as described (16) . Biotin signals were detected with FITC-avidin (Vector Laboratories) and amplified with one layer of biotinylated antiavidin antibody (Vector Laboratories), followed by one layer of FITC avidin (17) . Chromosomes and nuclei were counterstained with DAPI. The slides were examined with a Leica DMRBE microscope equipped with a cooled CCD camera (Hamamatsu 4800) and filter sets specific for the fluorochromes (DAPI, FITC, and Spectrum Orange).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of p53 Induction in MEF wt, p21-/-, or p19ARF-/- Cells.
We have shown previously that uptake of DNA from apoptotic bodies into normal cells results in cell cycle arrest and senescence. (7 , 8) . In contrast, DNA engulfed by phagocytosis is propagated in cells with inactivated p53. We used MEF cells originating from wt, p21-/-, or p19ARF-/- mice to investigate the potential role of these genes in mediating the p53-mediated senescence. First, we verified that these cell lines contained functional p53. The cells were exposed to {gamma} irradiation, and induction of p53 protein was detected by immunofluorescent staining analysis 3 h later. The cells were stained with a p53-specific mouse monoclonal antibody that showed a positive nuclear staining in the wt, p21-/-, or p19ARF-/- MEF cells (Fig. 1)Citation . MEFp53-/- cells were used as a negative control, and, as expected, no positive staining could be detected in these cells.



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  		Fig. 1. Induction of p53 in MEF p21-/- and MEF p19ARF cells. MEF p21-/-, p19ARF-/-, and MEF p53-/- cells were irradiated with 60 Gy. Induction of p53 protein was analyzed by immunofluorescence using the pAb421 anti-p53 antibody. Irradiated MEF p53-/- cells served as negative controls. Size bar = 20 µm.

 
REFrm Apoptotic Bodies Induce Focus Formation in MEF p21-/- Cells.
We then assessed whether apoptotic bodies derived from REFrm cells could induce focus formation in wt, p21-/-, or p19ARF-/- MEF cells. Apoptosis was induced in the donor REF or REFrm cells by {gamma} irradiation. The irradiated cells were then cocultured with the wt, p21-/-, or p19ARF-/- MEF recipient cells. Focus formation was scored in triplicate 10 days after the start of the experiment. As we have reported previously, no focus formation could be detected when apoptotic bodies derived from REFrm cells were added to wt MEF cells. However, foci were formed in MEF cells deficient in p21, as well as the MEF p53-/- cells, which served as a positive control (Fig. 2)Citation . No significant difference in the frequency of focus formation between the MEF p21-/- and the p53-/- cells could be detected. In contrast, no detectable focus formation was detected when p19ARF-/- cells were used as recipient cells. In addition, apoptotic bodies derived from REF cells did not induce any detectable focus formation in any of the recipient cell lines that were used.



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  		Fig. 2. Focus formation assay. Apoptosis was induced by {gamma} irradiation of REF or REFrm cells, which were subsequently added to cultures of MEF, MEF p53-/-, MEF p21-/-, and MEF p19ARF cells. Focus formation was scored after 10 days of coculture. The data reflect the mean value +/-SD of three independent experiments.

 
Stable Transfer and Expression of the hygr Gene in p21-/- MEF Cells.
The transfer of drug resistance genes is a useful method to assess the frequency of gene transfer and colony formation. Apoptosis was induced in REFrm cells transfected with the gene encoding hygromycin resistance or in REF cells as described. The resulting apoptotic bodies were then added to cultures of wt MEF, p53-/-, p21-/-, or p19ARF-/- MEF cells. Hygromycin selection of drug-resistant cells was started 48 h after the initiation of the experiment. The experiment was stopped 3 weeks later, and colonies were fixed and stained with Giemsa (Fig. 3A)Citation . The number of colonies per 10-cm Petri dish was counted, and the resulting data are shown in Fig. 3BCitation . Colony formation could only be detected in plates where REFrm apoptotic bodies had been added to either MEF p53-/- or p21-/- cells. No hygromycin-resistant cells could be detected in plates with MEF p19ARF-/- (Fig. 3B)Citation or wt MEF cells (data not shown).



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  		Fig. 3. Stable transfer of the gene encoding hygromycin resistance to MEF p21-/- cells. MEF cells with the indicated genetic backgrounds were cocultured with REF cells or REFrm cells transfected with the hygr plasmid. In A, colony formation in 10-cm Petri dishes was scored after 3 weeks of coculture by Giemsa staining. B, frequency of colony formation, shown as the average of three independent experiments.

 
Detection of the H-rasV12 and the Human c-myc Genes in Foci from MEF p21-/- Cells.
We next determined by PCR whether the genes encoding H-rasV12, human c-myc, or hygr could be detected in the recipient MEF p21-/- cells after coculture. Foci were isolated from plates with MEF p21-/- cultured with REFrm apoptotic bodies. DNA was isolated from a subset of the cells from these foci and subjected to PCR analysis. >90% of the isolated foci contained both the H-rasV12 and the human c-myc genes (Fig. 4, A and B)Citation . Cells harvested from these foci were propagated in vitro, and the maintenance of the transferred DNA was analyzed after 3 and 4 weeks of culture. After 3 weeks, approximately half of the cells was negative for H-rasV12 and human c-myc in the PCR analysis. At 4 weeks, no positive amplification of these genes could be detected in any of the cell lines. This shows that transferred DNA is gradually lost during propagation in vitro.



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  		Fig. 4. Detection of H-rasV12, human c-myc, or hygr genes in cells cocultivated with apoptotic bodies by PCR analysis. In A, PCR analysis showing the presence of the H-rasV12 or B. human c-myc genes in foci and the resulting cell lines after 3 and 4 weeks of propagation in culture. C, presence of the hygr gene in MEF p21-/- cells after coculture with apoptotic bodies derived from REFrm cells transfected with the hygr gene. M = 100-bp ladder.

 
Immediately after harvesting, ~40% of the foci also contained the hygr gene. We then assessed whether the hygr gene detected in these foci could be propagated in the presence of hygromycin selection. Positive amplification of the hygr gene could be detected at 3 and 4 weeks (Fig. 4C)Citation and was still detectable after 3 months in culture (data not shown). These findings indicate that transfer of the H-rasV12 and human c-myc oncogenes are not selected for in vitro. However, in the presence of strong selection pressure, genes, such as the hygr gene, can be maintained during cell proliferation.

REFrm x MEFp21-/- Foci Form Tumors in SCID Mice.
To assess the tumorigenicity of cells isolated from foci induced by REFrm apoptotic bodies, foci derived from REFrm x MEF p21-/- cocultures were injected into the s.c. space in SCID mice. In addition, an equivalent number of cells from REFrm x MEF p19ARF or REFrm x MEF cocultures was injected into parallel groups of animals. Four days after injection, tumors were palpable in mice injected with REFrm x MEF p21-/- cells. Tumor volume was measured every 3rd day with a caliper, and the mice in the REFrm x MEF p21-/- group were killed 22 days after injection when the tumors had reached a size of 2 cm in diameter (Fig. 5)Citation . No tumors were palpable in the animals injected with REFrm x MEF p19ARF-/- or REFrm x MEF cells, and these animals did not show any sign of tumor formation 6 months after injection (Table 1)Citation .



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  		Fig. 5. Tumor growth in SCID mice of MEF, MEF p21-/-, or p19ARF-/- cells cultured with apoptotic bodies derived from REFrm cells. Cells were cultured with REFrm apoptotic bodies in vitro and injected into the dorsal s.c. space of SCID mice. Cells (5 x 106) were injected into each animal (n = 7). The experiment was repeated three times. The graph shows data from one representative experiment.

 

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  		Table 1 Tumor formation in SCID mice

 
DNA and protein were isolated from five REFrm x MEF p21-/- tumors and analyzed by PCR for the presence of the H-rasV12 and c-myc genes or by protein expression. PCR analysis showed the presence of H-rasV12 and human c-myc DNA in all tumors analyzed (Fig. 6A)Citation . Active transcription of the detected DNA was indicated by the presence of the corresponding proteins that were detected with antibodies specific to H-rasV12 and human c-myc (Fig. 6B)Citation . This indicates that the H-rasV12 human c-myc oncogenes are selectively maintained in vivo during tumor growth, whereas in vitro, they are gradually lost. Next, we used FISH analysis to investigate how the transferred DNA was propagated in the REFrm x MEF p21-/- tumors. For this purpose, we used a chromosomal painting probe that hybridized specifically with rat genomic DNA, and no cross-hybridization to mouse DNA could be detected (Fig. 6C)Citation . The analysis showed the presence of rat chromosomes as well as fused rat-mouse chromosomes in the metaphase spreads REFrm x MEF p21-/- tumor cells (Fig. 6C)Citation .



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  		Fig. 6. Detection of the H-rasV12 and human c-myc genes in REFrm x MEF p21-/- tumors. In A, PCR analysis was used to detect the presence of H-rasV12 and the human c-myc genes in five individual REFrm x MEF p21-/- tumors. B, immunoprecipitation and Western blot analysis of H-rasV12 and human c-myc protein expression of the same tumors as shown in A. C, FISH analysis of rat DNA in metaphase spreads from REFrm x MEF p21-/- tumors. The FITC-labeled rat DNA painting probe (green) detects rat but not mouse DNA as shown in the controls. Rat chromosomes as well as rat and mouse hybrid chromosomes could be detected in metaphase spreads from REFrm x MEF p21-/- tumors. DNA was counterstained with the DAPI fluorochrome (blue).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we show that cells deficient in p21 are capable of replicating chromosomal DNA acquired by the uptake of apoptotic bodies. These data indicate that the p53 activation of p21 protects normal cells from propagating potentially pathological DNA.

In our experiments, we could not find any significant differences in the frequencies of focus and tumor formation or hygromycin resistance in the MEF p53-/- and MEF p21-/- cells. This indicates that p21 is the main effector of p53 protection against exogenous DNA. Several mechanisms can explain the p53-mediated activation of p21. One possible mechanism of activation of p53 is via the activation of p19ARF. It has been suggested that the normal role of the p19ARF protein is to respond to hyperproliferative growth. Indeed, several reports have shown that overexpression of Myc, E1A, or E2F1 in primary MEF cells rapidly induce ARF expression resulting in apoptosis via the activation of p53 (18 , 19) . Furthermore, expression of oncogenic ras in MEF cells leads to senescence (20) , and this senescent state has been shown to be dependent on the p19ARF pathway (21) . We hypothesized that the transfer of the H-rasV12 and myc genes could induce p53 via the activation of p19ARF. However, mouse fibroblasts deficient in p19ARF were unable to proliferate after uptake of apoptotic bodies. Another possible mechanism of p53 activation is via DNA damage. Our experiments indicate that large fragments of DNA are transferred via the uptake of apoptotic bodies. We suggest that double-stranded DNA breaks in the DNA from dying cells are being sensed as DNA damage by the recipient cells. This would trigger p53 and its downstream target genes via the DNA damage response pathways, such as the activation of the Ataxia-Telangiectasia-mutated kinase. The Ataxia-Telangiectasia-mutated kinase is activated by single or double DNA strand breaks and phosphorylates p53 at NH2-terminal sites, resulting in a block in the p53-MDM2 interaction (22) . The accumulation of p53 results in activation of transcription of the p21 gene, which mediates G1 arrest after {gamma} irradiation. The activation of p53-p21 pathway by DNA damage is independent of p19ARF as G1 arrest has been shown to be induced in p19ARF-/- MEF cells by irradiation (21) . This is consistent with our findings that transferred DNA is replicated in MEF p21-/- but not in p19ARF-/- cells.

The FISH analysis that specifically detected DNA derived from the apoptotic bodies showed that entire chromosomes or portions of chromosomes were present in the nuclei of the recipient MEF p21-/- cells (Fig. 6)Citation . Thus, the tumors consisted of mouse cells containing a variable number of rat chromosomes. Several independent groups have reported the findings of spontaneous cell hybrids in vivo (23) . These fusions have been shown to occur between tumor cells or between tumor cells and host cells. This is exemplified by the findings that in vivo fusion of the tumor cells and normal host cells resulted in the generation of a malignant phenotype (24 , 25) . It has therefore been proposed that the emergence of metastatic cell variants could arise as a consequence of tumor and host cell fusions (23) . Our data are consistent with the notion that the uptake of apoptotic bodies is a possible mechanism by which these spontaneously occurring hybrids are generated (26) . This mode of gene transfer is not restricted to fibroblasts because we have shown transfer DNA from apoptotic bodies to dendritic cells, macrophages, and endothelial cells (7 , 9) . It is therefore feasible that similar mechanisms may generate genetic diversity in other cell types that phagocytose apoptotic bodies, such as tumors of epithelial origin. The rate by which genes are exchanged will depend on the frequency of apoptosis in the tumor. Environmental factors, such as restricted access to blood circulation or hypoxia, both significantly increase apoptosis (27 , 28) . In addition, most tumor therapies, including chemo and radiation therapy, are aimed at reducing or eliminating tumor burden. Our studies support an additional effect of apoptosis, namely, generation of genetic diversity. This raises the question of additional and unwanted effects of apoptosis-based therapeutic strategies.


   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 Swedish Cancer Society and the Swedish Society of Medicine. Back

2 To whom requests for reprints should be addressed, at Cancer Center Karolinska Hospital, R8:03, S-171 76 Stockholm, Sweden. E-mail: lars.holmgren{at}cck.ki.se Back

3 The abbreviations used are: wt, wild type; MEF, mouse embryonic fibroblast; SCID, severe combined immune deficiency; REF, rat embryonic fibroblast; DAPI, 4',6-diamidino-2-phenylindole; RIPA, radioimmunoprecipitation assay; FISH, fluorescence in situ hybridization. Back

Received 8/10/01. Accepted 11/14/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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