Key Roles for E2F1 in Signaling p53-Dependent Apoptosis and in Cell Division within Developing Tumors

Molecular Cell, Vol. 2, 283–292, September, 1998, Copyright 1998 by Cell Press

Key Roles for E2F1 in Signaling p53-Dependent Apoptosis and in Cell Division within Developing Tumors Huichin Pan,*‡# Chaoying Yin,#* Nicholas J. Dyson,† Ed Harlow,† Lili Yamasaki,†§ and Terry Van Dyke*k * Department of Biochemistry and Biophysics University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599 † Massachusetts General Hospital Cancer Center Charlestown, Massachusetts 02129 ‡ Department of Life Sciences Chung Shan Medical and Dental College Taichung 402 Taiwan R. O. C. § Department of Biology Columbia University New York, NY 10027

Summary Apoptosis induced by the p53 tumor suppressor can attenuate cancer growth in preclinical animal models. Inactivation of the pRb proteins in mouse brain epithelium by the T 121 oncogene induces aberrant proliferation and p53-dependent apoptosis. p53 inactivation causes aggressive tumor growth due to an 85% reduction in apoptosis. Here, we show that E2F1 signals p53dependent apoptosis since E2F1 deficiency causes an 80% apoptosis reduction. E2F1 acts upstream of p53 since transcriptional activation of p53 target genes is also impaired. Yet, E2F1 deficiency does not accelerate tumor growth. Unlike normal cells, tumor cell proliferation is impaired without E2F1, counterbalancing the effect of apoptosis reduction. These studies may explain the apparent paradox that E2F1 can act as both an oncogene and a tumor suppressor in experimental systems. Introduction When activated by DNA damage, oncogenic events, oxidation stress, and other aberrant cellular states, p53 can induce apoptosis, an activity that has been linked to its tumor suppression capacity in animal models (Pan et al., 1997). The mechanism of p53-mediated apoptosis is poorly understood, and the molecular basis for p53 induction is not known. In previous work, we developed a transgenic mouse model, TgT121, in which epithelial brain tumors are initiated by inactivation of the pRb proteins with an N-terminal fragment of SV40 T antigen. Under these conditions, the normally nondividing choroid plexus (CP) cells are induced to proliferate, resulting in the induction of p53 target gene expression (Yin et al., 1997) and p53-dependent apoptosis (Symonds et al., 1994). The high apoptosis level is responsible for dramatic attenuation of tumor growth (Symonds et al., k To whom correspondence should be addressed (e-mail: tvdlab@

med.unc.edu). # These authors contributed equally to this work.

1994). Other systems, both cell culture and animals, have also demonstrated the induction of p53-dependent apoptosis upon inactivation of pRb or all pRb family proteins. p53-dependent apoptosis and tissue degeneration occurs in the lens upon expression of a truncated T antigen (Fromm et al., 1994) or HPV E7 (Pan and Griep, 1994) and in the retina upon expression of HPV E7 (Howes et al., 1994). Adenovirus E1A also causes p53dependent apoptosis in certain nontransformed cultured cells (Debbas and White, 1993; Lowe et al., 1994). Rb deficiency in mice results in extensive p53-dependent apoptosis in the embryonic central nervous system coincident with abnormal S phase entry of postmitotic neurons (Macleod et al., 1996) as well as in embryonic lens (Morgenbesser et al., 1994). In mouse embryonic fibroblasts (MEFs), p53 induction caused by some antineoplastic drugs is also pRb-dependent (Almasan et al., 1995). pRb itself is a tumor suppressor that is inactivated in a variety of cancers (Marshall, 1991; Weinberg, 1991). pRb is regulated during the cell cycle by phosphorylation controlled in part by G1 cyclin/cdks (Sherr, 1994; Weinberg, 1995). Roles for pRb in cell cycle regulation and terminal differentiation have been demonstrated (Lee et al., 1994; Zachsenhaus et al., 1996). At the molecular level, pRb appears to act by modulating the activity of specific transcription factors, the best studied of which is E2F1. Phosphorylation of pRb results in the activation of E2F1, which is thought to drive the G1 to S phase transition. Ectopic expression of E2F1 in quiescent mammalian cultured cells can drive S phase entry (Johnson et al., 1993) and can overcome G1 arrest induced by irradiation or inhibition of G1 CDK activity (DeGregori et al., 1995; Lukas et al., 1996). E2F1 activates the transcription of several S phase genes, such as dihydrofolate reductase, DNA polymerase a, and thymidine kinase, as well as several cell cycle genes including cyclin E, cyclin D, c-myc, and E2F itself (Hinds and Weinberg, 1994; Adams and Kaelin, 1995; Slansky and Farnham, 1996). However, the regulatory roles of E2F1 are complex, in that it can also act as a transcriptional repressor when complexed with Rb (Adams and Kaelin, 1995; Weintraub et al., 1995; Sellers et al., 1996). E2F1 was the first identified of a family of related factors, which now includes E2F1 through 5 (Sardet et al., 1995; Slansky and Farnham, 1996). The E2Fs require heterodimerization with DP1 or DP2 for transcriptional regulation and fall into two functional subgroups. E2Fs 1–3 efficiently induce S phase entry and preferentially bind pRb, while E2F4 and 5 associate with p107 or p130 and induce S phase weakly (Lees et al., 1993; Beijersbergen et al., 1994; Hijmans et al., 1995; DeGregori et al., 1997). Despite the evidence that E2F1 is involved in cell cycle regulation, however, development proceeds normally in E2F1-deficient mice (Field et al., 1996; Yamasaki et al., 1996). In addition to cell cycle activation, E2F1 overexpression has been shown to trigger apoptosis subsequent to S phase entry of quiescent cells (Qin et al., 1994; Shan and Lee, 1994; Wu and Levine, 1994; Kowalik et

Molecular Cell 284

al., 1995). The apoptosis is largely diminished in the absence of p53 function (Kowalik et al., 1995) and can be enhanced by exogenous p53 expression (Qin et al., 1994; Wu and Levine, 1994). Furthermore, E2F1 can induce endogenous p53 expression to trigger apoptosis in the presence of survival factors (Hiebert et al., 1995). Of the five known mammalian E2Fs, the ability to induce apoptosis is unique to E2F1 (DeGregori et al., 1997). In vivo, transgenic expression of Drosophila melanogaster dE2F in imaginal discs induces cell death along with DNA synthesis in some otherwise quiescent cells (Asano et al., 1996; Du et al., 1996). A role for E2F1 in apoptosis could explain the observation that mice deficient in E2F1, while developmentally normal, are prone to tumors with advanced age (Yamasaki et al., 1996). Indeed, thymocyte apoptosis at certain maturation stages is defective in E2F1 2/2 mice resulting in an overabundance of mature T cells (Field et al., 1996). However, a role for E2F1 in tumor suppression is difficult to reconcile with its apparent ability to drive the cell cycle and to act as an oncogene in cultured cells (Johnson et al., 1994; Xu et al., 1995). The role of E2F1 in p53dependent apoptosis in the context of tumor suppression has not previously been studied. Since E2F1-induced apoptosis in cultured cells is inhibitable through direct interaction with pRb (Shan et al., 1996; Hsieh et al., 1997), inactivation of pRb family function by DNA tumor virus proteins, such as T121, may induce apoptosis that is signaled by E2F1. To test this idea in vivo, we have examined the role(s) of E2F1 in p53dependent apoptosis and tumor growth in the TgT121 transgenic mouse model described above. In the brain tumors of these mice (deficient in pRb family function), p53-dependent apoptosis retards tumor growth, providing a model for simultaneously testing roles in p53dependent apoptosis and tumor suppression. Here, we examine tumor cell apoptosis and tumor growth in the presence and absence of E2F1 after interbreeding TgT 121 mice with E2F1-deficient mice (Yamasaki et al., 1996). We show that E2F1 has a clear role in the induction of p53-dependent apoptosis and, surprisingly, is also required for the tumor cell division cycle.

Results E2F1 Is Required for p53-Dependent Apoptosis TgT121 transgenic mice develop highly apoptotic slowgrowing brain tumors and die with a t50 of 26 weeks (Saenz-Robles et al., 1994; Symonds et al., 1994). The cells targeted in these mice, choroid plexus epithelium, normally withdraw from the cell cycle within 2 weeks after birth and remain in a resting state thereafter. However, in TgT121 mice these cells are induced to proliferate aberrantly by tissue-specific expression of T121, a truncated SV40 T antigen that inactivates the Rb family proteins but not p53. Tumor growth is suppressed in these mice by the induction of p53-dependent apoptosis. Inactivation of p53 causes dramatic acceleration of tumor growth due to an 85% reduction in apoptosis, and mice die rapidly with a t50 of 4 weeks (Symonds et al., 1994). T121-induced apoptosis depends on inactivation of pRb proteins, since mutation of this function abolishes both

CP cell proliferation and apoptosis (Saenz-Robles et al., 1994). To directly test whether E2F1 is required for the induction of p53-dependent apoptosis, we generated TgT121 mice deficient in E2F1 by a series of backcrosses with E2F12/2 mice, as described in Experimental Procedures. E2F1 is expressed in both transgenic and nontransgenic CP at approximately equal levels based on in situ RNA hybridization (data not shown). Apoptosis within the CP of several TgT121 E2F1 1/1, TgT121E2F1 1/ 2, and TgT121 E2F12/2 young mice (2–11 weeks) was measured in situ by TUNEL (Figure 1A). The apoptosis indices (AI) in control TgT121E2F11/1 and TgT121E2F1 1/ 2 littermates averaged 7.3 6 1.25% and 7.02 6 0.95%, respectively (Figure 1B). This level is similar to that previously determined for TgT121 (7.3 6 1.5%) (Symonds et al., 1994). However, in the absence of E2F1 (TgT121E2F1 2/2 mice) the AI was reduced to 1.79 6 0.31% (Figure 1B). This 80% decrease in apoptosis relative to TgT121E2F11/1 littermates is comparable, within experimental error, to the 85% apoptosis reduction previously observed upon p53 inactivation (Symonds et al., 1994; Figure 1C). These results show that E2F1 is required for p53-mediated apoptosis in this system and are consistent with the possibility that E2F1 functions upstream of p53 activation. E2F1 Acts Upstream of p53 Since p53-dependent apoptosis is induced in TgT121 CP by inactivation of the pRb family proteins, it follows that E2F1 could act upstream of p53. However, another possibility is that E2F1 acts in a parallel pathway that is also required. As an independent test of whether E2F1 acts in the p53 pathway, a p53 function that is not associated with CP apoptosis was assayed. We have shown that the p53 responsive gene, p21WAF1, is not involved in CP apoptosis (unpublished data). Yet, p21WAF1 expression is specifically induced by T121 and is strictly dependent on p53 (Figure 2, compare panels 2B and 2C). Thus, if E2F1 lies upstream of p53 activation, an E2F1 deficiency would cause a reduction in p53-dependent p21WAF1 expression as well as in apoptosis. Indeed, in the absence of E2F1, the level of p21WAF1 transcripts in CP was reduced by 89% (Figure 2D; Table 1). Furthermore, although the induction of p21WAF1 expression represents the best apoptosis-independent test of p53 function in this system, similar reductions in p53induced Mdm2 and Bax expression were also caused by E2F1 deficiency (Table 1). These results show that activation of multiple p53 functions (apoptosis and transactivation) requires E2F1 and indicate that E2F1 lies upstream of p53 (Figure 6). Impact of E2F1 Inactivation on Tumor Growth Upon p53 inactivation in TgT 121 mice, the 85% apoptosis reduction accelerates CP tumor growth (survival t50 5 4 weeks; Symonds et al., 1994; Figure 1C). Hence, a similar 80% apoptosis reduction in the absence of E2F1 may be expected to accelerate tumor growth. Yet, E2F1 appears not to be a target for inactivation in human tumors, and tumor development in E2F1-deficient mice occurs with long latency and in only 34% of the animals (Yamasaki et al., 1996).

E2F1 Role in Apoptosis and Tumorigenesis In Vivo 285

Figure 1. E2F-1 Is Required for p53-Dependent Apoptosis (A) Representative views of the in situ TUNEL assay in CP of TgT121E2F11/1 (a) and TgT121 E2F12/2 (b) mice. Apoptotic nuclei are stained brown and nonapoptotic nuclei are counterstained with methyl green (blue). Note the diminished apoptosis in CP of TgT121 E2F12/2 mice. (B) Apoptosis indices (AIs) within the CP of TgT121 mice (2–11 weeks). In each age group, one bar represents an average count of ten random CP fields for one mouse. Note that there is no significant difference in the AI of TgT121E2F1 1/1 and TgT121E2F1 1/2 CP. TgT121 E2F11/2 mice serve as a control in some sets. The average AI for all mice in the right panel is shown in the left panel. (C) The average relative AIs are compared for TgT121 mice with no deficiency and those with a deficiency in E2F1, p53, or Bax. The TgT121 value is 100%. The AI is reduced by 80% in the E2F1 2/2 background, 85% in the p532/2 background (Symonds et al., 1994), and 50% in the bax2/2 background (Yin et al., 1997). Bar, 25 mm.

To determine the overall impact of E2F1 deficiency on tumor growth, we compared the survival time and morphological development of CP tumors in TgT121 E2F11/1 and TgT121E2F12/2 mice. As previously shown, TgT121E2F11/1 mice developed slow-growing tumors, with a tumor survival t50 of 26 weeks. Death was not substantially accelerated in TgT121E2F1 2/2 mice (t50 5 20 weeks, Table 2). In addition, these mice died from hydrocephalus (excessive cerebral spinal fluid [CSF]) rather than tumor burden. The impact of E2F1 on tumor growth was independently evaluated by comparing the size of CP tumor masses in TgT121 mice with altered E2F1 genotypes at a variety of ages. A series of sections, spanning the entire brain and containing CP, was examined histologically. The tumor masses in TgT121E2F12/2 mice did not grow larger than those of TgT121 mice when followed for up to 11 weeks (prior to overt hydrocephalus; compare Figures 3B and 3E and 3C and 3F). In contrast, CP of TgT121p532/2 mice showed enlarged tumor masses by 4 weeks of age (compare Figures 3A and 3D). Thus, although p53-dependent apoptosis was severely diminished in the absence of E2F1, this did not lead to accelerated tumor growth as occurs upon p53 inactivation.

E2F1 Deficiency Interferes with the Tumor Cell Cycle Overall tumor growth rates are determined by the combination of cellular death and proliferation rates. Although E2F1 is clearly dispensable for most normal cell cycles, as evidenced by normal development and survival of E2F1 2/2 mice (Field et al., 1996; Yamasaki et al., 1996), it is possible that E2F1 is required for the aberrant S phase of tumor cells. To determine whether E2F1 has a role in the induction and maintenance of aberrant CP cell proliferation, the fraction of tumor cells in S phase was determined in the presence or absence of E2F1 by in situ immunodetection of BrdU incorporation (Figure 4A). TgT121E2F1 1/ 1 and TgT121E2F12/2 mice of different ages (6 to 25 weeks) were examined (Figure 4B). At all stages, a deficiency in E2F1 caused a clear reduction in the percentage of cells in S phase compared to controls (Figures 4B and 4C). By 6 weeks of age, there was a 42% reduction and by 25 weeks of age a 73% reduction in the tumor cell S phase fraction relative to TgT121 controls. On average, the S phase fraction was reduced by 61% in the absence of E2F1 (Table 2). Since a decrease in the number of BrdU positive cells could result from either reduced cell cycle activity or a

Molecular Cell 286

dramatic decrease in p53-dependent apoptosis and thus why E2F1 is not a tumor suppressor in this system.

Discussion Our results demonstrate the biological consequences of E2F1 inactivation to tumor development. Diminished apoptosis in TgT121E2F12/2 epithelial brain tumors indicates that E2F1 signals an oncogene-induced p53dependent apoptosis pathway. However, E2F1 deficiency does not accelerate tumor growth due to its requirement for efficient cell cycle activity. Thus, although p53-dependent apoptosis is defective in the absence of E2F1, any impact on tumor growth is counterbalanced by the accompanying reduction in tumor cell proliferation. Figure 2. E2F1 Is Upstream of p53 Activation CP from non-Tg (A), TgT121 (B), TgT121p532/2 (C), and TgT121E2F12/2 (D) mice was examined for p21WAF1 expression by in situ RNA hybridization with a p21WAF1 antisense RNA probe. The CP-containing region is framed with dotted lines. Expression of p21WAF1 is undetectable in nontransgenic CP (A) but dramatically induced upon expression of T121 (B). The expression is entirely dependent on p53 (C). This p53-dependent p21WAF1 expression is significantly reduced in the CP of TgT121E2F12/2 (D) mice. Quantification of p21WAF1 RNA in situ hybridization is shown in Table 1. CP, choroid plexus; Br, brain parenchyma surrounding choroid plexus. Bar, 200 mm.

shortened S phase, the percentage of M phase tumor cells was also determined. If cell proliferation is inhibited in the absence of E2F1, a reduction in the M phase percentage should mirror the observed reduction in S phase. M phase cells were detected by immunohistochemical staining of phosphorylated histone H3 (Hendzel et al., 1997). TgT 121E2F1 1/1 and TgT121E2F11/2 CP contained a similar percentage of M phase cells. However, without E2F1, there was a significant reduction in the fraction of cells in M (Figure 5A, compare panels 5Aa and 5Ab and 5Ac and 5Ad). Furthermore, when compared as a function of time, the relative percentage of cells in M, like those in S, decreased over time (Figure 5B). The parallel decrease in S and M phases demonstrates that E2F1 is required for efficient tumor cell proliferation. An increased effect with time indicates that the cell cycle is defective but not completely inactive. The observation that tumor cell proliferation is defective in the absence of E2F1 explains why overall tumor growth was not accelerated in TgT121E2F12/2 mice despite a

E2F1 Role in Signaling p53-Dependent Apoptosis Several in vitro studies have shown apoptosis induction associated with E2F1 activity (Qin et al., 1994; Shan and Lee, 1994; Wu and Levine, 1994; Kowalik et al., 1995; DeGregori et al., 1997), and now this study demonstrates that E2F1 is required for p53-dependent apoptosis in developing tumors. In the tumor model system described here, apoptosis is diminished by 80% upon E2F1 deficiency, similar to the 85% apoptosis reduction upon p53 inactivation (Table 2). This attenuation of p53dependent apoptosis is sufficiently large that E2F1 could be involved in inducing p53 activity. In the CP, an excellent measurement of p53 activity that is independent of the p53 apoptotic function (but entirely dependent on p53 activity) is the induction of p21WAF1 transcription. This functional test represents the best possible independent assessment of p53 activation in this tissue. Quantitative determination of p53 protein levels is difficult in CP, since this tissue comprises a small fraction (0.5%) of the brain. Moreover, p53 can be activated without significant level increases (Lutzker and Levine, 1996). We demonstrate here that E2F1 inactivation causes a nearly 90% reduction in p21WAF1 expression (Figure 2; Table 1). Inactivation of multiple p53 functions (apoptosis and apoptosis-independent transactivation) upon E2F1 deficiency indicates that E2F1 acts upstream of p53. It is unlikely that E2F1 directly induces p21WAF1 (Hiyama et al., 1997, 1998) in the CP. There is no induction of p21WAF1 in T121-expressing CP in the absence of p53, even though E2F1 is fully functional (Figure 2C). Furthermore, other unrelated p53 target genes, mdm-2

Table 1. E2F1 Is Required for p53 Target Gene Induction Relative % Transcript Levela Genes (Probes) WAF1

p21 mdm2 bax

Reduction in p53-Induced Level (%)

TgT121

TgT121p532/2

TgT121E2F12/2

TgT121E2F12/2

100 100 100

1.2 34 13

12 51 32

89 83 81

a Transcript levels were measured using radioactive in situ RNA hybridization on the mouse CP of indicated genotypes and quantified as described in Experimental Procedures. Absolute values of silver particle density in the CP of TgT121, TgT121p532/2, and TgT121E2F1 2/2 mice are 122.8, 1.5, and 14.7 for p21WAF1 ; 56.2, 19.1, and 29.0 for mdm2; and 94.8, 12.0, and 30.8 for bax, respectively. Relative transcript levels were determined by taking the value in TgT121 CP as 100%.

E2F1 Role in Apoptosis and Tumorigenesis In Vivo 287

Table 2. E2F1 Impact on Tumor Growth Genotype tg

Mutation

Relative % Apoptosisa

Relative % S Phaseb

Relative % M Phasec

CPE Neoplastic Growth

Survival (t50, weeks)

T121 T121 T121 T121 —

— p532/2 E2F1 1/2 E2F1 2/2 E2F1 2/2

100 14 6 10 89 6 13 21 6 5 0

100 109 ND 39 6 14 0

ND ND 100 32 6 17 0

slow rapid slow slow none

26 (n 5 16) 4 (n 5 4) 27 (n 5 21) 20d (n 5 22) .50e (n 5 5)

All numbers represent averages of several samples (see text) and have been rounded to the nearest integer. tg 5 transgene; ND 5 not determined. a Relative apoptosis index where the percentage of apoptotic cells in T121 CPE (7.3% on average) is considered 100%. b Relative percentage of S phase cells where the percentage of BrdU positive cells in T 121 CPE is considered 100%. c Relative percentage of M phase cells where the percentage of M phase cells (by phosphorylated histone H3 positivity) in T121E2F1 1/2 CPE is considered 100%. d Death in T 121 E2F1 2/2 mice was not due to tumor growth but to hydrocephalus, a condition caused by aberrant cerebral spinal fluid regulation, most likely secondary to CPE malfunction. e E2F2/2 mice develop a vareity of tumor types with 19%–34% of incidence around 18 months of age (Yamasaki et al., 1996).

and bax, are also similarly diminished in the absence of E2F1 (Table 1). Thus, these results are consistent with a model whereby T121 inactivates pRb resulting in the activation of E2F1 and the subsequent activation of p53 responses, including apoptosis (Figure 6). From our studies, it is not possible to determine how E2F1 might induce p53 activities. E2F1 could signal p53 directly via transcriptional regulation of p53 or of p53 regulatory factors. Alternatively, the signal could be indirect via the induction of S phase chromosomal activity by E2F1. The fact that apoptosis is diminished by 80% without E2F1, and yet there is still significant S phase activity (60% early in tumor growth), indicates that there may be a direct interaction between E2F1 and the p53 pathway. In support of this, other studies have shown that E2F1 transcriptional transactivation is not required for E2F1-

induced apoptosis (Hsieh et al., 1997; Philips et al., 1997). Furthermore, E2F2 and E2F3 expression in cultured cells induces S phase but not apoptosis or p53. Only E2F1 induces apoptosis and p53 accumulation in addition to S phase (DeGregori et al., 1997; Kowalik et al., 1998). Analysis of additional tumor types will be required to determine whether E2F1 functions broadly in tumorassociated apoptosis. However, two recent studies demonstrate a role for E2F1 in p53-dependent apoptosis during development. In the developing lens, transgenic expression of HPV E7, which also inactivates pRb proteins, induces p53-dependent apoptosis in association with proliferation (Pan and Griep, 1995). Upon E2F1 deficiency, both apoptosis and proliferation are substantially reduced (McCaffrey et al., submitted). p53dependent neuronal apoptosis in the developing central nervous system of Rb2/2 embryos (Macleod et al., 1996)

Figure 3. E2F1 Inactivation Does Not Accelerate Tumor Growth Morphology of CP tumor masses in lateral ventricles at similar positions is shown for TgT121E2F1 2/2 mice at 5 weeks (E) and 11 weeks (F) of age relative to their TgT121 littermates at the same age (B and C, respectively) in comparison with TgT121p532/2 (D) relative to TgT121 (A) at 4 weeks of age. Step section analysis was carried out to insure that appropriate size comparisons were made. Bar, 250 mm.

Molecular Cell 288

Figure 4. The Tumor Cell S Phase Is Defective without E2F1 (A) Shown are representative views of BrdU immunohistochemical staining within the CP of TgT121E2F1 1/1 (a and c) and TgT121E2F1 2/2 (b and d) mice at 6 weeks (a and b) and 11 weeks (c and d) of age. Red-stained nuclei have incorporated BrdU. The CP of nontransgenic mice beyond 2 weeks of age show no positive nuclei (data not shown). (B) The percentage of S phase cells (Experimental Procedures) in CP of TgT121 mice with altered E2F1 genotypes is shown. Each bar represents average counts of 10 fields in CP of a single mouse. At the left, the percentage of S phase cells is averaged for all mice of each genotype. (C) The percentage of TgT121E2F12/2 S phase cells relative to that of TgT121 littermates is shown as a function of time. Bar, 20 mm.

also requires E2F1 (Tsai et al., 1998). The same study shows that the p53-independent apoptosis in the peripheral nervous system is unaffected by the deficiency in E2F1. Together, these studies provide substantial evidence that E2F1 has a key role in p53-dependent apoptosis associated with aberrant cell cycle activity in vivo. E2F1 Role in the Tumor Cell Cycle In the present study, we show that along with a reduction in apoptosis, E2F1 deficiency also impairs S phase activity within epithelial brain tumors. This observation is especially interesting because E2F1 appears to be dispensable for most normal cell cycles in mammals. E2F1deficient mice develop normally and live long lives. Although a few tissues are anomalous (testicular atrophy, exocrine gland dysplasia [Yamasaki et al., 1996] and thymic enlargement [Field et al., 1996] are common), most cell cycle activity seems to proceed normally. However, our studies show that epithelial brain tumor cells clearly require E2F1 for efficient proliferation as evidenced by the average 60%–70% reduction in S and M phase cells, respectively (Table 2). The E2F1 requirement appears not to be a function of the cell type, since the CP tissue develops normally in E2F1-deficient mice.

Rather, the E2F1 role may be a function of the unscheduled nature of the proliferation induced by pRb inactivation. This is also true of pRb-deficient CNS neurons (Tsai et al., 1998) and lens fibroblasts (McCaffrey et al., submitted). In these aberrantly proliferating populations, E2F1 deficiency also causes a decrease in S phase cells, while development of the normal counterparts proceeds without effect in embryos deficient only in E2F1. E2F1 may be dispensable for normal cell cycles due to the availability of several additional E2Fs in mammals. Both E2F2 and E2F3 can induce S phase in cultured cells (Slansky and Farnham, 1996; DeGregori et al., 1997), and it’s possible that one or both of these factors regulates normal cell proliferation or can functionally compensate for the absence of E2F1. In Drosophila, unlike mammals, dE2F is required for normal development and its mutation causes lengthening of the embryonic S phase (Duronio et al., 1998). The presence of multiple E2Fs in mammals, however, does not overcome the requirement for E2F1 in aberrantly proliferating CP tumor cells (this paper), embryonic lens (McCaffrey et al., submitted), or CNS neurons (Tsai et al., 1998). The reason for this distinction in proliferating cells is not clear. Different circumstances for proliferation (e.g., pre- vs. postdifferentiation) could create distinct regulatory requirements.

E2F1 Role in Apoptosis and Tumorigenesis In Vivo 289

Figure 5. Optimal Tumor Cell Division Requires E2F1 (A) Mitosis is detected by immunohistochemical staining of the M phase-specific phosphorylated histone H3. Representative fields are shown for TgT121 (a and c) and TgT121 E2F2/2 (b and d) at 5 weeks (a and b), 23 weeks (c), and 26 weeks (d) of age. (B) The percentage of M phase cells in TgT121E2F1 2/2 CP relative to that in TgT121 E2F11/2 CP is shown as a function of time. Bar, 25 mm.

Alternatively, additional compensatory mechanisms (e.g., other E2Fs) may not be activated under the “aberrant” conditions produced by pRb deficiency. Our studies demonstrate that E2F1 has a significant impact on the overall tumor growth rate. In previous studies, we showed that the CP tumor growth rate is inversely correlated with the apoptosis level. That is, the higher the level of p53-dependent apoptosis, the slower the tumor growth and the longer survival time (Yin et al., 1997). For example, deficiency in Bax (a p53 regulated cell death inducer) (Miyashita and Reed, 1995) causes a 50% reduction in apoptosis and accelerates tumor growth 3-fold (Yin et al., 1997). Deficiency in p53 causes an 85% apoptosis reduction and a 7-fold increase in tumor growth rate (Symonds et al., 1994). In the absence of E2F1, apoptosis is inhibited by 80%, but tumor growth is not accelerated. We show here that this is because E2F1 is also required for efficient tumor cell proliferation. Since E2F1 is central to both the tumor cell cycle and to the ensuing cellular apoptotic defense, it possesses properties of both an oncogene and a tumor suppressor gene. This may explain the apparent paradox that E2F1 can act as an oncogene in cultured cell transformation, and yet E2F1-deficient mice are prone to tumorigenesis (see Introduction). The tumors that arise in E2F1 null mice (in a fraction of animals, at

late times) may reflect multiple mutations that overcome the E2F1-null antiproliferative effect. This would not happen in the natural course of tumorigenesis, which begins in an E2F1 positive background. E2F1 inactivation would not be selected for during tumorigenesis (as would the inactivation of other proapoptotic genes such as p53), because tumor cell proliferation would also be impaired. Likewise, E2F1 oncogene activation (without simultaneous inactivation of the p53 apoptotic pathway) would not occur due to the induction of extensive p53-dependent apoptosis. It will be important to determine whether E2F1 is required for tumor cell proliferation in diverse tumor types. E2F1 inactivation has been shown to attenuate pituitary and thyroid tumorigenesis in Rb 1/ 2 mice (Yamasaki et al., 1998). In an E2F1-deficient background, incidence of these tumors is reduced, and lifespan is significantly increased. Our studies now demonstrate a mechanism by which this occurs. Thus far, the requirement for E2F1 in aberrant proliferation has involved inactivation of pRb (or pRb, p107, and p130). An important issue is whether E2F1 is generally required for tumor cell proliferation or is specific to the inactivation of pRb proteins. Strikingly, most human tumors carry mutations that affect the known pRb pathway (Sherr, 1996). The studies presented here indicate that E2F1 could be an important

Molecular Cell 290

The following procedures were performed at room temperature unless otherwise stated. For BrdU immunostaining, mice were first injected intraperitoneally with BrdU labeling reagent (Amersham Cell Proliferation Kit) and sacrificed 1 hr later as previously described (Wu et al., 1996). Sections were incubated in 1 N HCl for 20 min and washed 3 times with PBS, followed by blocking with 10% normal horse serum (NHS) in PBS. Sections were then incubated with antiBrdU Mab 1 nuclease (Amersham Cell Proliferation Kit), for 1 hr. For M phase staining, sections were treated with trypsin (1 mg/ml) in PBS for 30 min at 378C, washed twice with PBS, and blocked in 5% normal goat serum in PBS. Sections were then incubated with an antibody (1:1000; rabbit polyclonal) against phosphorylated histone H3, which is specific for the M phase nuclei for 1 hr. After two washes in PBS following the primary Ab incubation, sections were incubated with biotin-conjugated secondary Ab (1:400 with 2% NHS in PBS) for 30 min followed by a 10 min wash with 0.1% Tween-20 in PBS. Immunocomplexes were detected using the Vectastain ABC alkaline phosphatase kit (Vector) according to the manufacturer’s instructions.

Figure 6. Model for E2F1 Role in CP Tumor Dynamics CP tumors are induced by the inactivation of one or more pRb proteins by their association with transgenically introduced T121. The inactivation of pRb proteins results in the activation of E2F1 and possibly other E2Fs. In this report, E2F1 is shown to be required for optimal S phase induction and tumor cell proliferation. Some S phase induction in the absence of E2F1 may be driven by other E2Fs (dashed arrow). E2F1 is also shown to be required for p53dependent apoptosis in CP and is proposed to lie upstream of p53 based on the reduction of p53-dependent apoptosis and transcriptional activation without E2F1. Dashed arrows above p53 activation indicate that these studies do not distinguish direct induction of the p53 pathway by E2F1 and indirect induction via other S phase activities (see text). Previous studies have shown that Bax is induced by p53 in this system and is required for much but not all of the apoptosis (Yin et al., 1997).

factor in the dynamics of these tumors and may be an appropriate target for specific drug therapy development. Experimental Procedures Generation of TgT 121 Transgenic Mice Defective at the E2F1 Locus Generation, screening, and characterization of TgT121 transgenic mice were described previously (Saenz-Robles et al., 1994; Symonds et al., 1994). These mice harbor the T 121 mutant T antigen gene under the control of the lymphotropic papovavirus (LPV) transcriptional signals (Chen et al., 1989). The T 121 encodes the first 121 amino acids of SV40 T antigen and is capable of binding to the pRb family proteins but not to p53. Mice harboring a homozygous null mutation at the E2F1 locus were described previously (Yamasaki et al., 1996). TgT121 transgenic mice heterozygous or homozygous for the E2F1 null allele were generated by crossing the TgT121 mice with the E2F1 2/2 mice through two generations. Genotypes were identified by PCR analysis of tail DNA as described (Symonds et al., 1994; Yamasaki et al., 1996).

In Situ Detection of Apoptosis Deparaffinized sections were treated with proteinase K (20 mg/ml) in PBS for 15 min at room temperature. Endogenous peroxidase activity was quenched with 2% H2 O2 in PBS for 5 min. Detection of apoptosis was performed using the in situ Apoptag kit (Oncor) according to the manufacturer’s protocol except that the sections were incubated with TdT for 15 min. Quantification of apoptosis was by counting 10 random fields of view at a magnification of 4003 per section assayed. Four experimental/control animal sets were analyzed. At least three different sections per animal were assayed, and the counts were averaged to obtain the apoptosis index. In Situ RNA Hybridization The pBluescript KS(1/2)-mp21WAF1 (Wu et al., 1996), the pBluescript KS(1/2)-mbax (Yin et al., 1997), and the pKG-mdm2 (a gift from Dr. Gigi Lozano) plasmids were used as templates for generating probes. The p21WAF1 antisense probe was generated by T7 transcription of the EcoRI-linearized template and the sense probe by T3 transcription of the XbaI-linearized template, the bax probes were generated as previously described (Yin et al., 1997), and the mdm2 antisense probe was generated by T7 transcription of the SalIlinearized template and the sense probe by T7 transcription of the SmaI-linearized template. Probes were radioactive-labeled using [a-35S]UTP and were used at a concentration of 5 3 10 4 cpm/ml. The slides were hybridized as described (Pan and Griep, 1995; Yin et al., 1997) except that autoradiography was carried out at 48C for 3 days for p21WAF1 and for 1 week for bax and mdm-2. Signal densities of RNA hybridization were quantified using NIH image 1.58. The threshold was set such that signal in the negative control (sense probe) was just detectable. Means were determined from 10 random areas of CP for each sample. Acknowledgments This work is dedicated to the memory of my (T. V. D.) mother, Betty Van Dyke, who died of lung cancer while it was in progress. Her inspiration will never be forgotten. We appreciated the technical support of Tracy Bartolotta, Kitti Ballenger, Le Zhang, John Kim, Chris Hoecke, and Lyn Rodgers, and the administrative support of Linda Greene. Tammy Bowman performed some initial mouse crosses. Thanks to Bob Duronio for critical reading of the manuscript and for helpful suggestions. We thank Tyler Jacks and Anne Griep for communicating results from their laboratories prior to publication. This work was supported by grants to T. V. D. from the National Cancer Institute (CA46283 and CA65773). Received May 14, 1998; revised July 20, 1998.

Histology and Immunohistochemistry Brain tissues were fixed in 10% formalin (Sigma) overnight, washed in dH 2O followed by 70% ethanol, and embedded in paraffin. 6 m sections were deparaffinized using Hemo-De (Fisher) followed by dehydration in graded ethanols. For histology, sections were stained with hematoxylin and eosin as described (Saenz-Robles et al., 1994).

References Adams, P.D., and Kaelin, W.G., Jr. (1995). Transcriptional control by E2F. Semin. Cancer Biol. 6(2), 99–108. Almasan, A., Yin, Y., Kelley, R.E., Lee, E.Y.H.P., Bradley, A., Li, W.,

E2F1 Role in Apoptosis and Tumorigenesis In Vivo 291

Bertino, J., and Wahl, G.M. (1995). Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2Fresponsive genes, and apoptosis. Proc. Natl. Acad. Sci. USA 92(12), 5436–5440. Asano, M., Nevins, J.R., and Wharton, R.P. (1996). Ectopic E2F expression induces S phase and apoptosis in Drosophila imaginal discs. Genes Dev. 10, 1422–1432. Beijersbergen, R.L., Kerkhoven, R.M., Zhu, L., Carlee, L., Voorhoeve, P.M., and Bernards, R. (1994). E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo. Genes Dev. 8(22), 2680–2690. Chen, J., Neilson, K., and Van Dyke, T. (1989). Lymphotropic papovavirus early region is specifically regulated in transgenic mice and efficiently induces neoplasia. J. Virol. 63, 2204–2214. Debbas, M., and White, E. (1993). Wild-type p53 mediates apoptosis by E1A, which is inhibited by E1B. Genes Dev. 7, 546–554. DeGregori, J., Kowalik, T., and Nevin, J.R. (1995). Cellular targets for activation by the E2F1 transcription factor include DNA synthesis and G1/S-regulatory genes. Mol. Cell. Biol. 15, 4215–4224. DeGregori, J., Leone, G., Miron, A., Jakoi, L., and Nevins, J.R. (1997). Distinct roles for E2F proteins in cell growth control including a unique role for the E2F1 protein as a signal for apoptosis. Proc. Natl. Acad. Sci. USA 94, 7245–7250. Du, W., Xie, J-E., and Dyson, N. (1996). Ectopic expression of dE2F and dDP induces cell proliferation and death in the Drosophila eye. EMBO J. 15, 3684–3692. Duronio, R.J., Bonnette, P.C., and O’Farrell, P.H. (1998). Mutations of the Drosophila dDP, dE2F, and cyclin E genes reveal distinct roles for the E2F-DP transcription factor and cyclin E during the G1-S transition. Mol. Cell. Biol. 18, 141–151. Field, S.J., Tsai, F.Y., Kuo, F., Zubiaga, A.M., Kaelin, W.G., Jr., Livingston, D.M., Orkin, S.H., and Greenberg, M.E. (1996). E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85, 549–561. Fromm, L., Shawlot, W., Gunning, K., Butel, J., and Overbeek, P. (1994). The retinoblastoma protein binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice. Mol. Cell. Biol. 14(10), 6743–6754. Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli, T., Brinkley, B.R., Bazett-Jones, D.P., and Allis, C.D. (1997). Mitosisspecific phosphorylation of histone H3 initiates primarily within peicentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360. Hiebert, S.W., Packham, D.K., Strom, D.K., Haffner, R., Oren, M., Zambetti, G., and Cleveland, J.L. (1995). E2F1:DP1 induced p53 and overrides survival factors to trigger apoptosis. Mol. Cell. Biol. 15, 6864–6874. Hijmans, E.M., Voorhoeve, P.M., Beijersbergen, R.L., Van’T Veer, L.J., and Bernards, R. (1995). E2F-5, a new E2F family member that interacts with p130 in vivo. Mol. Cell. Biol. 15(6), 3082–3089. Hinds, P.W., and Weinberg, R.A. (1994). Tumor suppressor genes. Curr. Biol. 4, 135–141. Hiyama, H., Iavarone, A., LaBaer, J., and Reeves, S.A. (1997). Regulated ectopic expression of cyclin D1 induces transcriptional activation of the cdk inhibitor p21 gene without altering cell cycle progression. Oncogene 14, 2533–2542. Hiyama, H., Iavarone, A., and Reeves, S.A. (1998). Regulation of the cdk inhibitor p21 gene during cell cycle progression is under the control of the transcription factor E2F. Oncogene 16, 1513–1523. Howes, K.A., Ransom, N., Papermaster, D.S., Lasudry, J.G.H., Albert, D.M., and Windle, J.J. (1994). Apoptosis or retinoblastoma: alternative fates of photoreceptors expressing the HPV-16 E7 gene in the presence or absence of p53. Genes Dev. 8, 1300–1310. Hsieh, J.-K., Fredersdorf, S., Tony, K., Martin, K., and Lu, X. (1997). E2F1-induced apoptosis required DNA binding but not transactivation and is inhibited by the retinoblastoma protein through direct interaction. Genes Dev. 11, 1840–1852. Johnson, D.G., Schwarz, J.K., Cress, W.D., and Nevins, J.R. (1993). Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365, 349–352.

Johnson, D.G., Cress, W.D., Jakoi, L., and Nevins, J.R. (1994). Oncogenic capacity of the E2F1 gene. Proc. Natl. Acad. Sci. USA 91, 12823–12827. Kowalik, T.F., DeGregori, J., Schwarz, J.K., and Nevins, J.R. (1995). E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis. J. Virol. 69(4), 2491–2500. Kowalik, T.F., DeGregori, J., Leone, G., Jakoi, L., and Nevins, J.R. (1998). E2F1-specific induction of apoptosis and p53 accumulation, which is blocked by Mdm2. Cell Growth Differ. 9, 113–118. Lee, E., Hu, N., Yuan, S., Cox, L., Bradley, A., Lee, W., and Herrup, K. (1994). Dual roles of the retinoblastoma protein in cell cycle regulation and neuron differentiation. Genes Dev. 8, 2008–2021. Lees, J.A., Satio, M., Vidal, M., Valentine, M., Look, T., Harlow, E., Dyson, N., and Helin, K. (1993). The retinoblastoma protein binds to a family of E2F transcription factors. Mol. Cell. Biol. 13, 7813–7825. Lowe, S., Jacks, T., Housman, D., and Ruley, H. (1994). Abrogation of oncogene-associated apoptosis allows transformation of p53deficient cells. Proc. Natl. Acad. Sci. USA 91, 2026–2030. Lukas, J., Petersen, B.O., Holm, K., Bartek, J., and Helin, K. (1996). Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression. Mol. Cell. Biol. 16, 1047–1057. Lutzker, S.G., and Levine, A.J. (1996). A functionally inactive p53 protein in teratocarcinoma cells is activated by either DNA damage or cellular differentiation. Nat. Med. 2, 804–810. Macleod, K.F., Hu, Y., and Jacks, T. (1996). Loss of Rb activates both p53-dependent and independent cell death pathways in the developing mouse nervous system. EMBO J. 15(22), 6178–6188. Marshall, C.J. (1991). Tumor suppressor genes. Cell 64, 313–326. Miyashita, T., and Reed, J.C. (1995). Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80, 293–299. Morgenbesser, S., Williams, B., Jacks, T., and DePinho, R. (1994). p53-dependent apoptosis produced by Rb-deficiency in the developing mouse lens. Nature 371, 72–74. Pan, H., and Griep, A.E. (1994). Altered cell cycle regulation in the lens of HPV-16 E6 or E7 transgenic mice: implications for tumor suppressor gene function in development. Genes Dev. 8, 1285–1299. Pan, H., and Griep, A.E. (1995). Temporally distinct patterns of p53dependent and p53-independent apoptosis during mouse lens development. Genes Dev. 9, 2157–2169. Pan, H., Yin, C., and Van Dyke, T. (1997). Apoptosis and cancer mechanisms. Cancer Surv. 29, 305–327. Philips, A.C., Bates, S., Ryan, K.M., Helin, K., and Vousden, K.H. (1997). Induction of DNA synthesis and apoptosis are separable functions of E2F-1. Genes Dev. 11, 1853–1863. Qin, X.Q., Livingston, D.M., Kaelin, W.G., and Adams, P.D. (1994). Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediatied apoptosis. Proc. Natl. Acad. Sci. USA 91, 10918–10922. Saenz-Robles, M.T., Symonds, H., Chen, J., and Van Dyke, T. (1994). Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen. Mol. Cell. Biol. 14 (4), 2686–2698. Sardet, C., Vidal, M., Cobrinik, D., Geng, Y., Onufryk, C., Chen, A., and Weinberg, R.A. (1995). E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle. Proc. Natl. Acad. Sci. USA 92(6), 2403–2407. Sellers, W., Rodgers, J., and Kaelin, W. (1996). A potent transrepression domain in the retinablastoma protein induces a cell cycle arrest when bound to E2F sites. Proc. Natl. Acad. Sci. USA 92, 11544– 11548. Shan, B., and Lee, W. (1994). Deregulated expression of E2F-1 induces S-phase entry and leads to apoptosis. Mol. Cell. Biol. 14, 8166–8173. Shan, B., Durfee, T., and Lee, W.H. (1996). Disruption of RB/EBF-1 interaction by single point mutations in E2F-1 enhances S-phase entry and apoptosis. Proc. Natl. Acad. Sci. USA 93, 679–684. Sherr, C. (1994). The ins and outs of RB: coupling gene expression to the cell cycle clock. Trends. Cell Biol. 4, 15–18.

Molecular Cell 292

Sherr, C.J. (1996). Cancer cell cycles. Science 274, 1672–1677. Slansky, J.E., and Farnham, P.J. (1996). Introduction to the E2F family: protein structure and gene regulation. Curr. Top. Microbiol. Immunol. 208, 1–30. Symonds, H., Krall, L., Remington, L., Saenz-Robles, M., Lowe, S., Jacks, T., and Van Dyke, T. (1994). p53-dependent apoptosis suppresses tumor growth and progression in vivo. Cell 78, 703–711. Tsai, K.Y., Hu, Y., Macleod, K.F., Crowley, D., Yamasaki, L., and Jacks, T. (1998). Mutation of E2F1 suppresses apoptosis and inappropriate S-phase entry and extends survival of Rb-deficient mouse embryos. Mol. Cell 2, 293–304. Weinberg, R.A. (1991). Tumor suppressor genes. Science 254, 1138– 1146. Weinberg, R.A. (1995). The retinoblastoma protein and cell cycle control. Cell 81, 323–330. Weintraub, S.J., Chow, K.N., Luo, R.X., Zhang, S.H., He, S., and Dean, D.C. (1995). Mechanism of active transcriptional repression by the retinoblastoma protein. Nature 375, 812–815. Wu, H., Wade, M., Krall, L., Grisham, J., Xiong, Y., and Van Dyke, T. (1996). Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development, and regeneration. Genes Dev. 10, 245–260. Wu, X., and Levine, A.J. (1994). p53 and E2F-1 cooperate to mediate apoptosis. Proc. Natl. Acad. Sci. USA 91, 3602–3606. Xu, G., Livingston, D.M., and Krek, W. (1995). Multiple members of the E2F transcription factor family are the products of oncogenes. Proc. Natl. Acad. Sci. USA 92(5), 1357–1361. Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E., and Dyson, N.J. (1996). Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85, 537–548. Yamasaki, L., Bronson, R., Williams, B.O., Dyson, N.J., Harlow, E., and Jacks, T. (1998). Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1(1/2)mice. Nat. Genet. 18, 360–362. Yin, C., Knuson, C.M., Korsmyers, J.S, and Van Dyke, T. (1997). Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature 385, 637–640. Zachsenhaus, E., Jiang, Z., Chung, D., Marth, J.D., and Philips, R.A. (1996). pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis. Genes Dev. 10, 3051–3064.