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Cell Growth & Differentiation Vol. 10, 147-154, March 1999
© 1999 American Association for Cancer Research

Overproduction of MDM2 in Vivo Disrupts S Phase Independent of E2F11

Valerie Reinke, Donna M. Bortner, Lisa L. Amelse, Karen Lundgren2, Michael P. Rosenberg, Cathy A. Finlay and Guillermina Lozano3

Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [V. R., L. L. A., G. L.]; and Glaxo Wellcome Research and Development, Research Triangle Park, North Carolina 27709 [D. M. B., K. L., M. P. R., C. A. F.]


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Expression of a ß-lactoglobulin (BLG)/mdm2 transgene (BLGmdm2) in the epithelial cells of the mouse mammary gland causes an uncoupling of S phase from M phase, resulting in polyploidy and tumor formation. The cell cycle defects are independent of interactions with p53. Because MDM2 also binds and activates the S phase-specific transcription factor E2F1, we hypothesized that increased E2F1 activity causes the development of the BLGmdm2 phenotype. We, therefore, generated BLGmdm2 mice that were null for E2F1. We observed no notable differences in histology or cyclin gene expression between BLGmdm2 and BLGmdm2/E2F1-/- mice, indicating that endogenous E2F1 activity was not required for the BLGmdm2 phenotype. Because, depending on the experimental system, either loss of E2F1 function or overexpression of E2F1 results in transformation, we also tested whether overexpression of E2F1 augmented the severity of the BLGmdm2 phenotype by generating mice that were bitransgenic for BLGmdm2 and BLGE2F1. We observed a unique mixture of the two single transgenic phenotypes histologically and found no significant changes in cyclin levels, indicating that overexpression of E2F1 had no effect on the BLGmdm2 transgenic phenotype. Thus, increased expression or absence of E2F1 does not affect the ability of MDM2 to disrupt the cell cycle.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The most well understood function of the protein encoded by the mdm2 oncogene is its ability to bind and inactivate the p53 tumor suppressor (1) . mdm2 is also a transcriptional target of p53 (2 , 3) , and it thereby completes a feedback loop for the regulation of p53 activity. Genetic experiments in the mouse have illustrated the functionally significant relationship of these two molecules. The early embryonic lethality of mice lacking mdm2 is completely rescued by deletion of p53 (4 , 5) . These data demonstrate that MDM2 is both necessary and sufficient to regulate p53 activity in early mouse development. However, these experiments cannot address possible additional functions of MDM2 because the mdm2-deficient mouse dies at 5.5 days of embryogenesis.

Other lines of evidence indicate that MDM2 does, indeed, have a function in addition to regulation of p53. Most human tumors with an amplified mdm2 gene retain a wild-type p53, indicating that overexpression of MDM2 is sufficient to abrogate p53 activity (6) . However, tumors that contain both a p53 mutation and mdm2 amplification have been identified and are associated with a worse prognosis than are tumors with a single alteration, indicating that a dual mutation can provide an additional growth advantage (7) . Additionally, overexpression of MDM2 in tissue culture cells that lack p53 confers a transformation phenotype on those cells (8) .

Finally, and most convincingly, overexpression of mdm2 in vivo leads to disruption of the normal cell cycle, independent of p53 (9) . In these experiments, the BLG4 promoter, which is active only in the pregnant and lactating mammary gland (10) , was used to drive expression of an mdm2 transgene (BLGmdm2) in the mouse. Mammary glands from lactating female transgenic mice displayed abnormal development, marked histologically by the presence of abnormally large, polyploid epithelial cells. Additionally, these epithelial cells still synthesized DNA at a time when the cells of a normal gland would have ceased to proliferate and been fully differentiated. Most remarkably, this phenotype occurred even in a p53-/- background, indicating that increased MDM2 levels can affect cell proliferation and differentiation in this tissue independent of p53.

The mechanism by which MDM2 overproduction disrupts the coordination of DNA synthesis in S phase and cytokinesis is unknown. However, one possible model involves the ability of MDM2 to bind and stimulate the activity of the S-phase transcription factor E2F1/DP1 (Fig. 1Citation ; Ref. 11 ). During the G1 phase of the cell cycle, the tumor suppressor Rb binds and inhibits E2F1 activity (12, 13, 14) . E2F1 is released as a function of Rb phosphorylation by cyclin-dependent kinases late in G1 and becomes transcriptionally active (15 , 16) . E2F1/DP1 then activates the expression of a number of genes involved in S phase, such as cyclin E (17) , dihydrofolate reductase (18 , 19) , thymidine kinase (20) , and DNA Pol {alpha} (17 , 21) . E2F1 is a potent facilitator of DNA synthesis, as demonstrated by the driving of quiescent cells into S phase by overexpression of E2F1 alone (22, 23, 24) .



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  		Fig. 1. Model for MDM2 involvement in the Rb/E2F1 pathway. MDM2 might prevent E2F1 function during G1-phase transcriptional repression when associated with Rb, influence the Rb/E2F1 interaction itself, or promote the ability of free E2F1 to activate S-phase gene transcription.

 
The creation of mice deficient for E2F1 provides support for an opposing function of E2F1. Mice lacking E2F1 are predisposed to hyperplasia and tumor development in certain tissues (25 , 26) . These data indicate that E2F1 can actually act as a negative growth regulator in vivo. The molecular mechanism for this activity is unknown but has been attributed to the fact that E2F1 can promote p53-dependent apoptosis (23 , 24 , 27) . Lack of p53-dependent apoptosis in E2F1-deficient mice (26) might accelerate tumor formation. Additionally, E2F1, when complexed with Rb during the G1 phase of the cell cycle, can mediate the transcriptional repression of S-phase genes (28, 29, 30, 31) . E2F1, thus, provides both positive and negative control of S-phase entry.

Because MDM2 binds E2F1 and stimulates its activity in S phase, we asked whether this interaction was responsible for the cell cycle defects induced by MDM2 overexpression in mammary epithelial cells. To test in vivo the hypothesis that the BLGmdm2 phenotype was due to increased E2F1-mediated transactivation of S-phase genes, we generated mice carrying the BLGmdm2 transgene in an E2F1 null background. Histological analysis and examination of S phase demonstrated that E2F1 is not required for the development of the BLGmdm2 phenotype. In addition, we generated bitransgenic mice by crossing mice that overexpress a BLGE2F1 transgene in mammary epithelial cells, which causes hyperplasia and increased apoptosis, with BLGmdm2 mice. The mammary glands in mice overexpressing both the E2F1 and mdm2 transgenes showed a combination of the individual phenotypes histologically and did not demonstrate a significant change in the amount of inappropriate DNA synthesis. The above experiments define an alternative pathway for MDM2 function in vivo, in addition to known interactions with p53 and E2F1.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Increased Cyclin A but not Cyclin D or E Expression in BLGmdm2 Mammary Tissue.
The phenotype of the BLGmdm2 transgenic mouse was initially identified as a lack of cellular proliferation in the mammary gland during pregnancy and lactation (9) . Additionally, a large percentage of the cells in the gland had enlarged, often multiple, nuclei and were polyploid. This phenotype was most apparent at midlactation, a time when the wild-type gland has already completed proliferation and differentiation and is at full capacity for milk production. In contrast, the BLGmdm2 transgenic gland produced little milk, and the epithelial cells also displayed signs of continued DNA synthesis, as determined by BrdUrd incorporation (Fig. 2)Citation . To elucidate the molecular nature of the transgenic phenotype, we asked whether there were any alterations in the production of various cell cycle-regulated proteins at different stages of mammary gland development. Because cyclin expression is tightly regulated in various phases of the cell cycle, we focused on cyclin D1 (G1), cyclin E (G1-S), cyclin A1 (S-G2), and cyclin B1 (G2-M). To that end, we performed immunohistochemistry on wild-type and BLGmdm2 transgenic mammary glands at midpregnancy and midlactation, using antibodies raised against the various cyclins.



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  		Fig. 2. Analysis of cell cycle gene expression in wild-type (WT) or BLGmdm2 transgenic mammary glands. Wild-type and BLGmdm2 mammary glands at day 10 of lactation were stained with H&E and photographed at x200 magnification. Immunohistochemical analysis was performed on wild-type or BLGmdm2 mammary glands to measure the levels of BrdUrd and cyclins D, E, and A. All immunohistochemical analysis was performed on mammary glands taken at day 10 of lactation, with the addition of samples at day 14 of gestation for cyclin D and cyclin A. Microscopy for all immunohistochemistry was performed at x400 magnification.

 
Two G1 cyclins tested, cyclin D1 and cyclin E, showed similar production patterns in both wild-type and transgenic mammary glands. Specifically, at day 14 of pregnancy, when growth factors were stimulating cyclin D1 production (15) , mammary epithelial cells from both wild-type and transgenic mice were positive for cyclin D1 (Fig. 2)Citation . This expression decreased somewhat in animals of either genotype by day 10 of lactation, when the cells were no longer receiving proliferation signals from the extracellular matrix (Fig. 2)Citation . Cyclin E, a G1-S phase cyclin, was produced in most cells of the mammary gland of either genotype at day 14 of gestation (data not shown) or at day 10 of lactation (Fig. 2)Citation .

Immunohistochemistry performed with an antibody to the S-G2-specific cyclin A revealed an interesting difference between the wild-type and the BLGmdm2 transgenic mammary gland. Cyclin A was detectable in both tissues during pregnancy, but during lactation, only the BLGmdm2 transgenic gland continued to be positive for cyclin A (Fig. 2)Citation .

Two different cyclin B antibodies were used to measure the levels of cyclin B in the epithelial cells of the BLGmdm2 transgenic mice. These antibodies only weakly stained a positive control. Therefore, we stained mammary epithelial cells of BLGmdm2 transgenic mice with two other antibodies for mitosis specific proteins, namely, MPM-2 and histone H3 phosphorylation (32 , 33) . Both antibodies positively stained the mammary epithelial cells of BLGmdm2 transgenic mice at day 10 of lactation (Fig. 3)Citation . The histone H3 phosphorylation antibody was comparable to the control sample at day 18 of gestation, whereas the MPM-2 antibody was weaker compared to day 14 of gestation, when most of the mammary epithelial cells are cycling. These data suggest that the cells of the mammary gland continue to make mitosis-specific proteins at a time point when they should be fully differentiated.



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  		Fig. 3. Analysis of mitotic gene expression in wild-type (WT) or BLGmdm2 transgenic mammary glands. Epithelial cells of the mammary glands of the BLGmdm2 transgenic mice were stained with antibodies to histone H3 phosphorylation (H3P) or MPM-2. Negative normal controls (blank) and BLGmdm2 sections were taken at day 10 of lactation. Positive controls were from day 14 (MPM-2) or 18 (H3P) of gestation.

 
BLGmdm2 and BLGmdm2/E2F1-/- Mammary Glands Are Histologically Similar.
Mice carrying the BLGmdm2 transgene demonstrate a p53-independent function for MDM2. Specifically, in the presence or absence of p53, the mammary epithelial cells of BLGmdm2 transgenic mice are hypoproliferative and become large, multinucleate, and polyploid. Because E2F1 is bound and activated by MDM2 in tissue culture (11) , we examined whether E2F1 contributed to the transgenic phenotype (see Fig. 1Citation for model). BLGmdm2 mice were crossed with mice heterozygous for a null allele of E2F1 (25) . BLGmdm2/E2F1+/- F1 progeny were then back-crossed to E2F1+/- mice. The resulting BLGmdm2/E2F1-/- females were mated and sacrificed at day 10 of lactation. The mammary glands of these mice were then compared with the mammary glands of wild-type, BLGmdm2, and E2F1-/- mice to determine whether there were histological differences, changes in cyclin gene expression, and BrdUrd incorporation, which shows the extent of DNA synthesis.

The general hypoplasia and enlarged or multiple nuclei present in BLGmdm2 transgenic mice were also present in BLGmdm2 transgenic E2F1 null mice but not in wild-type or E2F1 null mice (Fig. 4)Citation . Additionally, the extent of DNA synthesis was roughly equivalent in tissues carrying the BLGmdm2 transgene regardless of the E2F1 genotype (Fig. 4Citation and Table 1Citation ). In contrast, the wild-type and E2F1-/- cells of the mammary glands, which are histologically similar, showed virtually no BrdUrd incorporation (Fig. 4Citation and Table 1Citation ). Investigation into changes in expression of cyclin genes yielded no differences between genotypes, with the exception of cyclin A. As in the BLGmdm2 tissue, cyclin A was produced in some but not all of the BLGmdm2/E2F1-/- cells, whereas there was no appreciable cyclin A production in the wild-type and E2F1-/- cells (Fig. 4)Citation . Surprisingly, cyclin E levels were not decreased in the absence of E2F1, as might be expected of an E2F1 transcriptional target (Fig. 4Citation ; Ref. 17 ). Because cyclin A overexpression has been associated with apoptosis (34) , we also used terminal deoxytransferase-mediated dUTP-biotin nick end labeling to measure apoptosis. BLGmdm2/E2F1-/- mammary epithelial cells were terminal deoxytransferase-mediated dUTP-biotin nick end labeling negative, as were BLGmdm2 transgenic mice (9) . These data demonstrated that there was no significant difference between BLGmdm2 transgenic glands in the presence or absence of E2F1.



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  		Fig. 4. Histological comparison between BLGmdm2 and BLGmdm2/E2F1-/- mammary glands. Mammary glands were taken at day 10 of lactation from mice with the following genotypes: wild-type (WT), BLGmdm2, E2F1-/-, and BLGmdm2/E2F1-/-. The glands were then fixed, embedded, and sectioned. Immunohistochemical analysis for BrdUrd, cyclin E, and cyclin A was performed on sections of glands from mice of each genotype and photographed at x400 magnification.

 

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  		Table 1 Quantitation of BrdUrd incorporationa

 
Overexpression of E2F1 Does Not Alter the Effects of MDM2 Overproduction.
To extend the above observations, we further tested the hypothesis of E2F1 involvement in the BLGmdm2 phenotype. If the ability of E2F1 to promote S phase is increased in the presence of MDM2, then simultaneous overproduction of E2F1 and MDM2 should exacerbate the BLGmdm2 phenotype. To test this possibility, we used mice that overexpress an E2F1 transgene in the mammary gland (BLGE2F1) and display a hyperproliferation of cells and increased apoptosis.5

To create mice that overproduce both E2F1 and MDM2, we crossed mice carrying the BLGE2F1 transgene with BLGmdm2 transgenic mice. Females carrying one allele of each transgene were sacrificed 10 days after giving birth, and the mammary glands in these bitransgenic mice were compared with those from mice carrying either of the two single transgenes. Histological examination of H&E-stained sections indicated that the bitransgenic mammary tissue had a unique mixture of the two single transgenic phenotypes (Fig. 5)Citation . Both small, hyperproliferative cells, indicative of E2F1 overproduction, and large, multinucleate cells, indicative of MDM2 overproduction, were apparent. Additionally, inappropriate DNA synthesis, as measured by BrdUrd incorporation, was present in bitransgenic mammary tissue at levels similar to those of either single transgenic gland (Fig. 5Citation and Table 1Citation ). No changes in the levels of cyclins A or E were observed in the bitransgenic compared to the single transgenic glands (Fig. 5)Citation , although increased cyclin E might be expected in the glands of mice carrying the BLGE2F1 transgene. Thus, these data indicate that increased levels of E2F1 did not alter the phenotype of BLGmdm2 transgenic glands and further support the finding that E2F1 function is not required for the cell cycle defects in BLGmdm2 transgenic mammary glands.



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  		Fig. 5. Histological comparison between BLGE2F1 and BLGmdm2/BLGE2F1 mammary glands. Mammary glands taken from mice transgenic for either BLGE2F1 or BLGmdm2/BLGE2F1 at day 10 of lactation were fixed, embedded, and sectioned. H&E staining as well as immunohistochemical analysis for BrdUrd, cyclin E, and cyclin A were performed. H&E sections were photographed at x200 magnification, and all sections for immunohistochemical analysis were photographed at x400 magnification. Arrows highlight the small hyperproliferative cells indicative of E2F1 overproduction (black arrow) and the large multinucleate cells indicative of MDM2 overproduction (white arrow).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Transgenic mice overproducing MDM2 exhibit a disruption of normal cellular proliferation in the mammary gland, in a p53-independent manner (9) . This phenotype is marked by a decrease in mammary epithelial cell number, and the cells tend to contain enlarged or multiple nuclei that undergo multiple rounds of inappropriate DNA synthesis and can become polyploid. A molecular explanation for this phenotype is difficult to formulate because very little is known about MDM2 function outside of its ability to inhibit p53 function. The first step in further characterization of the BLGmdm2 phenotype was to identify the defective cell cycle stage by investigating cyclin gene expression, which pinpointed S phase as a likely candidate.

Analysis of cyclin gene expression in the mammary epithelial cells of wild-type mice in comparison to the BLGmdm2 transgenic mice only revealed significant differences in cyclin A levels. Cyclin A was produced at high levels in many cells of the transgenic mammary gland but not the wild-type gland. These data reinforce the notion that DNA synthesis is aberrant in the MDM2-overexpressing cells and indicate that the cells are either stranded in S phase, perhaps because they are unable to recognize an exit signal, or are hypersensitive to signals which cause them to enter S phase inappropriately. Interestingly, a transgenic mouse overexpressing cyclin A in the mammary gland has been generated using the same BLG promoter used in this study (34) . The overexpression of cyclin A caused nuclear abnormalities such as multinucleation and karyomegaly and an increased number of apoptotic cells as compared to normal mammary epithelial cells. The absence of apoptosis in the mammary epithelial cells of the BLGmdm2 transgenic mice described here, even in the presence of high levels of cyclin A, may be due to the ability of MDM2 to inhibit p53-dependent apoptosis (35) .

Rather surprisingly, cyclin E was present in virtually every epithelial cell of either wild-type or transgenic glands, possibly because the cells are arrested (or trying to arrest, in the case of the BLGmdm2 transgenic gland) at the G1 phase of the cell cycle or because cyclin E levels do not noticeably oscillate in this tissue.

Several proteins besides p53 have been identified that interact with MDM2. MDM2 binds p19ARF, one of the proteins encoded by the INK4a locus (36, 37, 38) . Because this interaction causes the degradation of MDM2, it is unlikely to be responsible for the phenotype of the BLGmdm2 mice, which is due to the overproduction of MDM2. MDM2 has more recently been shown to bind Numb, a protein that participates in cell fate specification (39) , but the functional significance of this interaction is not clear. The interaction of MDM2 with two other proteins, Rb and E2F1, could, however, result in the BLGmdm2 phenotype. MDM2 can bind Rb and ultimately decrease its ability to arrest cells in G1 in tissue culture (40) . Although it would be of interest to test genetically the relevance of this interaction in generating the BLGmdm2 phenotype, the Rb-/- mouse dies during embryogenesis (41 , 42) , and there are no available mice overproducing Rb in the appropriate tissue. MDM2 can also bind E2F1/DP1 and increase its ability to activate transcription of S-phase genes in tissue culture cells (11) . We, therefore, tested the possibility of an MDM2 interaction with E2F1 as the likely factor in creating the BLGmdm2 phenotype. By crossing BLGmdm2 mice with mice either lacking or overexpressing E2F1, we were able to determine that there was no requirement for E2F1 in the BLGmdm2 phenotype.

MDM2 could regulate E2F1 function by a variety of mechanisms (see Fig. 1Citation for model). For example, it could lift the Rb/E2F1-mediated transcriptional repression of S-phase genes, it could encourage dissociation of Rb from E2F1, or it could act directly with E2F1 to activate S-phase genes. The ultimate result of all of these activities would be the promotion of S phase, one of the key findings in the mammary gland of mice overproducing MDM2. Therefore, we considered it quite possible that MDM2 was acting on E2F1 to cause inappropriate DNA synthesis. However, no changes were identified in BLGmdm2 mice in the presence or absence of E2F1, indicating that the promiscuous activation of endogenous E2F1 by MDM2 was not a critical factor in the development of the BLGmdm2 phenotype. Due to the multiplicity of E2F transcription factors, it is possible that endogenous E2F1 is not an essential regulator of the cell cycle in the epithelial cells of the mammary gland but that a different family member performs such a function.

Because E2F1 appears to have dual functions and too little or too much E2F1 can lead to cell cycle defects, we also tested whether overproduction of E2F1 in conjunction with MDM2 could exacerbate the phenotype of BLGmdm2 transgenic mice. We tested this possibility in vivo by generating mice that overproduce both MDM2 and E2F1. Again, we did not see an increase in the severity of the BLGmdm2 phenotype, nor did we see any major differences in the BLGE2F1 phenotype (see Fig. 5Citation ). Instead, we saw a unique mixture of the two phenotypes. This observation is interesting because it demonstrates that neither protein is more dominant than the other one. These data, in combination with the lack of alterations of the BLGmdm2 phenotype in either the absence or overexpression of E2F1 described in this report, lead to the conclusion that these two cell cycle regulators act independently of each other in this tissue.

Although we have focused our discussion on MDM2-interacting proteins, the possibility also exists that it is the ability of MDM2 to function as a transcription factor that results in the BLGmdm2 phenotype. MDM2 has many characteristics of transcription factors, including an acidic region, a nuclear localization signal, and a RING finger (43) , and it can activate transcription when fused to the Lex A DNA-binding domain (44) . Thus, the overexpression of mdm2 in the mouse mammary gland may result in the alteration of gene expression.

MDM2, therefore, appears to be acting through a novel mechanism, when it is overproduced in the mammary gland of mice during pregnancy and lactation, to induce the phenotype of hypoproliferation, unregulated DNA synthesis, and polyploidy. Our findings effectively rule out interactions with both p53 and E2F1 as causes. What remains to be defined is the pathway by which MDM2 disrupts the normal proliferation and development of the mammary gland. Experiments addressing potential MDM2 transcriptional regulation or identifying novel MDM2-interacting proteins could possibly clarify this currently undefined pathway.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Mouse Breeding and Genotyping.
BLGmdm2 transgenic mice (line TG3640; Ref. 9 ) were maintained as hemizygotes by crossing to C57Bl/6J wild-type mice. 129/Sv-C57BL/6 mice heterozygous for a mutant E2F1 allele (E2F1+/-) were obtained from L. Yamasaki (University of Columbia, New York, NY; Ref. 25 ) and crossed with mice carrying the BLGmdm2 transgene. BLGmdm2/E2F1+/- progeny were back-crossed with E2F+/- mice to generate offspring that were BLGmdm2/E2F1-/-. BLGE2F1 transgenic mice (line TG3604) were crossed with BLGmdm2 mice (line TG3640) to create mice that carried both transgenes in a hemizygous state.

The presence of the BLGmdm2 transgene was determined by a dominant coat color marker that had been coinjected with the transgene and by PCR with transgene-specific primers BLG and MDM2, as described previously (9) . E2F1 heterozygous and homozygous mutant animals were identified by PCR, as described previously (25) , with the following exceptions. PCR was performed for both wild-type and mutant alleles together using 16 pmol each of L26 and L28 primers and 32 pmol of L31 primer per reaction. The PCRs (25 µl) were amplified using AmpliTaq (Perkin-Elmer) for 1 cycle (94°C 5 min), 35 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min), and 1 cycle (72°C for 7 min). BLGE2F1 transgenic mice were identified by D. M. B.5

Nulliparous females of the appropriate genotypes were mated and sacrificed at day 14 or 18 of gestation or at day 10 of lactation. If necessary, pups born to BLGmdm2 females were removed to a foster mother and replaced with slightly older pups to allow for continued nursing. Most animals were given an i.p. injection of 100 µg of BrdUrd in PBS per g of body weight {approx}2 h before sacrifice. The first abdominal (number 4) mammary glands on both sides of the animal were dissected for analysis.

Immunohistochemistry.
Mammary gland tissues were fixed in 0.4% paraformaldehyde in PBS at 4°C overnight, washed twice in PBS, and dehydrated through a graded series of ethanols, from 70 to 100%, according to standard procedures. The tissue was then incubated in xylene for 30 min before it was embedded in paraffin. Sections were cut at 7 µm and placed on lysine-coated slides. After rehydration, the slides were incubated in 0.01 M citrate buffer (pH 7.0) in a steamer for 25 min for antigen retrieval and then soaked in 0.3% hydrogen peroxide in methanol for 15 min. After rinsing in PBS, the slides were blocked with serum from the Vectastain kit (Vector Laboratories, Burlingame, CA) and then incubated with the appropriate antibody for 1 h at 37°C in a humidified chamber. MDM2 (9312) and cyclin E (1014) antibodies were rabbit polyclonal antisera raised by our laboratory and used at a 1:250 dilution. Cyclin A (C-19; polyclonal; 1:250) and cyclin D1 (72–13G; monoclonal; 1:100) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cyclin B (Santa Cruz Biotechnology and Upstate Biotechnology, Inc.), MPM-2 (Upstate Biotechnology, Inc.), and H3P (a gift from David Allis, University of Virginia Health Sciences Center, Charlottesville, VA) were also used at 1:100, 1:200, and 1:500 dilutions, respectively. All immunohistochemistry was performed with the Elite Vectastain Kit for mouse or rabbit (Vector Laboratories) according to the manufacturer’s instructions. Staining was detected with the substrate DAB (Vector Laboratories). All immunostained slides were counterstained with Nuclear Fast Red (Vector Laboratories) before dehydrating and mounting with Permount. BrdUrd immunostaining was performed using the BrdUrd staining kit (Zymed, San Francisco, CA) according to the manufacturer’s instructions.


   Acknowledgments
 
We thank Lili Yamasaki for the E2F1 null mouse and for helpful discussions.


   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 This work was supported in part by United States Army Medical Research and Materiel Command Grant DAMD17-96-6222 (to G. L.). Back

2 Present address: Agouron Pharmaceuticals, Inc., San Diego, CA 92121. Back

3 To whom requests for reprints should be addressed, at The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-8945; Fax: (713) 794-4295; E-mail: gglozano{at}notes.mdacc.tmc.edu Back

4 The abbreviations listed are: BLG, ß-lactoglobulin; BrdUrd, bromodeoxyuridine. Back

5 D. M. Bortner, unpublished observations. Back

Received for publication 8/25/98. Revision received 1/20/99. Accepted for publication 1/21/99.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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