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Am J Physiol Renal Physiol 293: F52-F59, 2007. First published April 25, 2007; doi:10.1152/ajprenal.00119.2007
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EDITORIAL FOCUS

Involvement of the CDK2-E2F1 pathway in cisplatin cytotoxicity in vitro and in vivo

Fang Yu, Judit Megyesi, Robert L. Safirstein, and Peter M. Price

Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Submitted 9 March 2007 ; accepted in final form 18 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
E2F1 is a key regulator that links cell cycle progression and cell death. E2F1 activity is controlled by Cdk2-cyclin complexes via several mechanisms, such as phosphorylation of retinoblastoma protein (pRb) to release E2F1, direct phosphorylation, and stable physical interaction. We have demonstrated that cisplatin cytotoxicity depends on Cdk2 activity, and Cdk2 inhibition protects kidney cells from cisplatin-induced cell death in vitro and in vivo. Now we show that E2F1 is an important downstream effector of Cdk2 that accumulates in mouse kidneys and in cultured mouse proximal tubular cells (TKPTS) after cisplatin exposure by a Cdk2-dependent mechanism. Direct inhibition of E2F1 by transduction with adenoviruses expressing an E2F1-binding protein (TopBP1) protected TKPTS cells from cisplatin-induced apoptosis, whereas overexpression of E2F1 caused cell death. Moreover, E2F1 knockout mice were markedly protected against cisplatin nephrotoxicity by both functional and histological criteria. Collectively, cisplatin-induced cell death is dependent on Cdk2 activity, which is at least partly through the Cdk2-E2F1 pathway both in vitro and in vivo.

acute kidney injury; cell cycle; cell death

CISPLATIN IS WIDELY USED to treat several types of solid tumors in cancer patients (9). Its clinical utility is limited by its nephrotoxicity, to which renal proximal tubule epithelial cells are especially sensitive (37, 42). Multiple mechanisms are implicated in cisplatin-induced nephrotoxicity, which results in tubular cell death by both apoptosis and necrosis (16, 30, 32, 53) and in acute loss of kidney function.

We and other groups showed in vivo that many normally quiescent kidney cells enter the cell cycle after cisplatin administration, as indicated by an increase in nuclear proliferating cell nuclear antigen (PCNA) levels, as well as [3H]thymidine and bromodeoxyuridine (BrdU) incorporation into nuclear DNA (30, 31). Coincident with increased cell cycle activity, cisplatin administration induces upregulation of p21, a cyclin-dependent kinase (Cdk) inhibitor (31). This protein is a positive effector on the fate of cisplatin-exposed renal tubule cells in vivo and in vitro (30, 38), and we reported that the mechanism of p21 protection is by direct inhibition of Cdk2 activity (51). Recently, we demonstrated that cisplatin cytotoxicity depends on Cdk2 activity, and Cdk2 inhibition protected kidney cells from cisplatin-induced cell death in vitro and in vivo (39). The mechanism of Cdk2 involvement in cell death is the subject of this report. Cdk2 is a kinase participating in cell cycle progression and is an upstream regulator of E2F1. E2F1 plays an important role in coordinating events connected with both cell cycle progression and cell death. This led us to hypothesize that E2F1 may participate in the Cdk2-dependent cell death pathway(s) induced by cisplatin.

We show here that E2F1 accumulation correlated with cisplatin exposure and required Cdk2 activity both in cultured mouse proximal tubular cells (TKPTS) and in mouse kidneys. Direct inhibition of E2F1 protected TKPTS cells from cisplatin-induced apoptosis whereas overexpression of E2F1 by adenoviral vectors caused cell death. Moreover, E2F1 knockout mice were used to demonstrate that E2F1 deficiency ameliorated the functional and morphological consequences of cisplatin-induced acute kidney injury. Taken together, our data demonstrate that E2F1 functions downstream of Cdk2 activity to participate in cisplatin cytotoxicity. Thus cisplatin-induced cell death depends on Cdk2 activity, which is at least partly through the Cdk2-E2F1 pathway in vitro and in vivo.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and Administration of Drugs

Experiments were performed on 10- to 12-wk-old male mice that weighed 22–28 g. E2F1 knockout mice (B6;129S4-E2F1–/–) (8) and their wild-type controls (B6;129/SF2/J) were purchased from Jackson Laboratory. Wild-type 129Sv mice were also used. The mice were maintained on a standard diet, and water was available freely. Cisplatin was administered by a single intraperitoneal injection of 20 mg/kg, a dosage that produces severe acute kidney injury in mice (31). Some mice (wild-type 129Sv background) also received daily intravenous injections of purvalanol (Tocris Cookson) as previously described (39). Animals were killed painlessly with methods of euthanasia approved by the Panel on Euthanasia of the American Veterinary Medical Association. The induction of acute kidney injury was monitored by following urea nitrogen levels in serum (BUN) and creatinine concentration in serum that were obtained by retroorbital bleeding using commercial kits (Biotron Diagnostics and Sigma Diagnostics, respectively).

Morphological Assessment

Three days after cisplatin treatment, kidneys were removed, immersed in 4% neutral-buffered formaldehyde, and fixed for 72 h. The tissues were paraffin embedded and processed for light microscopy. Sections of 5 µm were stained with hematoxylin-eosin, and histological criteria were determined. The following parameters were chosen as indicative of morphological damage to the kidney after cisplatin injection: tubular necrosis, brush border loss, tubular cast formation, presence of neutrophils, edema, tubular dilatation, distal tubule damage, red blood cell extravasation, and tubular degeneration. These parameters were evaluated as described previously (30) on a scale from 0 to 4: not present (0), mild (1), moderate (2), severe (3), and very severe (4). Each parameter was determined on at least five different animals by a histologist who was blinded to the source of the sections. Statistical significance was assessed by the two-sided t-test for independent samples, and P < 0.05 was considered significant.

Cell Culture

Cells were grown at 37°C in 5% CO2. TKPTS cells (7) were cultured in DMEM+Ham's F-12 medium supplemented with 50 µU/ml insulin and 7% FBS. Cisplatin was added to cultures, when indicated, to a final concentration of 25 µM when cells were ~75% confluent, and cells were grown for an additional 24 h. Adenoviruses expressing dominant-negative Cdk2 (DN-Cdk2) or topoisomerase IIbeta binding protein 1 (TopBP1) (27) were added to a final multiplicity of infection (MOI) of 100 18 h before cisplatin. Purvalanol was dissolved in DMSO and added 4 h before cisplatin to a final concentration of 9 µM. Adenoviruses expressing E2F1 (27) were added to 100 MOI, and where indicated 9 µM purvalanol was added at the same time as Ad-E2F1. In addition, adenoviruses expressing green fluorescence protein (GFP) were used as control.

Cell Death Determination

Fluorescence-activated cell sorter analysis. Cells were harvested by trypsinization and collected by centrifugation (10 min, 500 g). Cell pellets were resuspended in 0.3 ml of PBS that contained 5 mM EDTA, and 0.7 ml of ethanol was added. Cells were incubated at 4°C for 16 h, collected by centrifugation (10 min, 2,000 rpm), and resuspended in 0.5 ml of PBS-EDTA. RNase A was added (50 µl, 10 mg/ml), and the suspension was incubated at 25°C for 30 min. Propidium iodide (PI) was added (450 µl, 100 µg/ml), and samples were analyzed by using FACSCalibur (Becton Dickinson). At least 10,000 cells were analyzed for each culture condition, and values were based on three separate cultures. The percentage of cells in sub-G1/G0 (apoptotic fraction), G1/G0, S, and G2/M phases was determined by using a cell-cycle analysis program (WinMDI 2.8; Scripps Institute). Cells in the subdiploid (sub-G1/G0) region of the histogram are classified as apoptotic as previously reported (4, 38, 51). These analyses were performed on at least four separate cell cultures.

4',6'-Diamidino-2-phenylindole staining. Cells grown on coverslips were washed with PBS and fixed for 5 min with neutral-buffered formaldehyde. Fixed cells were stained with 4',6'-diamidino-2-phenylindole (DAPI; VECTOR) for 10 min. Fluorescence images were taken using a fluorescence microscope (Nikon Eclipse E800) with a DAPI filter and a x100 oil objective.

Trypan blue extrusion assay. Cells were stained with 0.2% trypan blue (Sigma-Aldrich) for 5 min. Cells unable to extrude trypan blue were considered nonviable, whereas unstained cells were considered viable. The stained and nonstained cells were counted separately on a hemocytometer using an inverted microscope (Nikon Eclipse TE200). The number of stained cells, expressed as a percentage of total cells, was used as a measure of cell death.

Adenoviruses

Adenoviruses expressing DN-Cdk2, TopBP1, and E2F1 were purified by CsCl banding as described previously (39, 51).

Western Blot Analysis

Proteins were extracted from TKPTS cells using a lysis buffer that contained 50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 0.5% NP-40, and 1% Triton X-100 with phosphatase inhibitor cocktail I and II, and proteinase inhibitor (Sigma-Aldrich). Western blot analyses were as described previously (38, 51). In brief, protein concentration was determined by using a Bio-Rad protein assay. Samples (protein, 100 µg/lane) from TKPTS cells or from nuclear extracts of kidneys (see immediately below) were boiled and electrophoresed using 12% SDS-PAGE (23) and transferred to polyvinylidene difluoride membranes. After being blocked with 5% nonfat dry milk in TBST, the membrane was incubated at 4°C overnight with primary antibody. After being washed, horseradish peroxidase-conjugated secondary antibody (anti-mouse or anti-rabbit) was applied (Amersham Biosciences). Proteins that bound to the secondary antibody were visualized by using ECL (Amersham Biosciences). The primary antibodies included an E2F1 (C20) polyclonal antibody (Santa Cruz Biotechnology), cdc2 p34 monoclonal antibody (Santa Cruz Biotechnology), and beta-actin monoclonal antibody (Sigma-Aldrich).

Kinase Assay for Cdk2

For assay of in vivo activity, kidneys were homogenized with a Teflon glass homogenizer in buffer that contained 10 mM HEPES (pH 7.6), 25 mM KCl, 1 mM EDTA, 10% glycerol, 1.8 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM dithiothreitol, and phosphatase and proteinase inhibitors as described above. The homogenate was layered over 2.2 ml of this buffer, and nuclei were pelleted by centrifugation at 24,000 rpm in an SW60 rotor. Nuclei from two mouse kidneys were resuspended in 1 ml of cold lysis buffer (as discussed immediately above), sonicated, and centrifuged for 10 min in an Eppendorf centrifuge. The supernatant was used for histone H1 kinase activity. Kinase activity of Cdk2 was assayed by a modified histone H1 kinase assay (11, 21, 39). Briefly, protein extracts (200 µg) were immunoprecipitated by agarose-immobilized anti-Cdk2 antibody (Santa Cruz Biotechnology) for 4 h at 4°C with constant rocking and washed three times with lysis buffer and once with kinase buffer that contained 20 mM Tris·HCl (pH 7.4), 10 mM MgCl2, and 1 mM dithiothreitol. Agarose beads were resuspended in 20 µl of kinase buffer that contained 2 µg of histone H1 (Upstate Biotechnology), 20 µM ATP, and 10 µCi of [{gamma}-32P]ATP (Amersham Biosciences) and incubated for 30 min at 30°C. The reaction was stopped by Laemmli buffer (23). Samples were boiled and electrophoresed by 12% SDS-PAGE as described above and autoradiographed.

Statistical Analyses

Statistical analysis was performed with ANOVA and a t-test. Results were expressed as means ± SE. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Induction of E2F1 Accumulation in Response to Cisplatin In Vitro and In Vivo Requires Cdk2

It has been reported previously that the accumulation of E2F1 protein level is a critical parameter to its apoptotic property (13, 24, 25, 46). To investigate whether E2F1 was involved in cisplatin cytotoxicity, we first examined the accumulation of E2F1 protein following treatment with cisplatin both in vitro and in vivo. E2F1 is induced following cisplatin treatment in cultured TKPTS cells (Fig. 1A). Also, E2F1 protein level was increased 1 and 2 days after cisplatin injection in mouse kidney extracts (Fig. 1B, bottom). We recently showed the correlation of Cdk2 activity with cell death after cisplatin exposure and that inhibition of Cdk2 activity before cisplatin treatment protected kidney tubular cells in vitro and in vivo (39). In cultured TKPTS cells, inhibition of Cdk2 by DN-Cdk2 expression adenoviruses or by purvalanol before cisplatin treatment also blocked the cisplatin-induced accumulation of E2F1, which correlates with the increase in Cdk2 activity (Fig. 1A). Cells not treated with cisplatin transduced with DN-Cdk2 adenoviruses or treated with purvalanol had no effect on E2F1 protein level (data not shown). Correspondingly, Cdk1, an E2F1 target gene, was increased in response to the accumulation of E2F1 by cisplatin exposure, indicating the increase in E2F1 transcription activity. However, in the absence of Cdk2 activity, cisplatin failed to induce E2F1 accumulation and transcription activity, as indicated by the normal level of Cdk1. In vivo, as shown in Fig. 1B, accumulation of E2F1 (middle and bottom) corresponded to the increase in Cdk2 activity (top), whereas inhibition of Cdk2 activity by purvalanol before cisplatin exposure prevented the induction of E2F1. These results correlate an increase of E2F1 with cisplatin-induced cell death in vivo and in vitro, and confirm that it is induced downstream of Cdk2.


Figure 1
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  		Fig. 1. Cdk2 activity is correlated with the induction of E2F1 accumulation after cisplatin exposure in vitro and in vivo. A: induction of E2F1 accumulation in vitro requires Cdk2 activity after cisplatin exposure. Proteins from treated TKPTS cells were subjected to kinase assay of Cdk2 activity on histone H1 (top) and Western blotting with antibodies against E2F1, beta-actin, and Cdk1. B: E2F1 protein accumulation with an increase in Cdk2 activity in response to cisplatin in vivo. Kinase assay of Cdk2 activity on histone H1 (top) and Western blotting was performed to detect E2F1 protein level (middle and bottom, respectively) in mouse kidney nuclei after cisplatin treatment. Mice either were not treated with cisplatin (Control) or were treated with 20 mg/kg cisplatin for 24 (1D Cisplatin) or 48 h (2D Cisplatin). In addition, some mice received daily intravenous injections of purvalanol at 30 mg/kg as described in MATERIALS AND METHODS. The bottom panel is a protein density analysis of induction of E2F1, and values represent means ± SE from 5 mice.

 
Protection of TKPTS Cells from Cisplatin-Induced Apoptosis by E2F1 Inhibition

We next determined whether inhibition of E2F1 can protect cultured TKPTS cells from cisplatin-induced apoptosis. Recent reports showed that TopBP1 (DNA topoisomerase II beta binding protein 1) specifically inhibited multiple activities of E2F1 via direct protein-protein interaction (27, 28). As shown in Fig. 2A, transduction of cells with TopBP1 expression adenoviruses protected from cisplatin-induced apoptosis. Cultured TKPTS in the absence of cisplatin had a background level of apoptosis (1.88 ± 0.49%) as determined by fluorescence-activated cell sorter analysis (Fig. 2B). Transduction with TopBP1-adenovirus had no effect on cell viability (2.19 ± 0.31%). Administration of cisplatin in the absence of E2F1 inhibition by TopBP1 increased apoptosis to 20.04 ± 4.19% of the cells. However, addition of TopBP1-adenovirus 18 h before cisplatin exposure significantly reduced the level of apoptosis to 4.81 ± 1.04%. Thus the data indicated that E2F1 inhibition significantly reduced death signaling during cisplatin cytotoxicity.


Figure 2
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  		Fig. 2. Protection of cultured mouse proximal tubular cells (TKPTS) from cisplatin-induced apoptosis by E2F1 inhibition. A: fluorescence-activated cell sorter analysis (FACS) analysis of PI-stained ethanol-fixed TKPTS cells: untreated (top left), treated with 100 multiplicity of infection (MOI) of topoisomerase IIbeta binding protein 1 (TopBP1)-adenovirus (bottom left), 25 µM cisplatin (top right), or pretreated with TopBP1-adenovirus for 18 h followed by cisplatin exposure for 24 h (bottom right). At least 10,000 cells were analyzed for each culture condition. B: bar graph of FACS analyses for apoptosis (percentage of cells in sub-G1/G0 fraction). Values are means ± SE from 4 separate cultures that were treated the same as described in A.

 
Expression of E2F1 Induces Cell Death in TKPTS Cells, Which Requires Cdk2

We demonstrated that E2F1 death signaling is a component of cisplatin cytotoxicity, and next investigated whether E2F1 induction alone is sufficient to induce cell death. We therefore tested the effect of E2F1 overexpression on TKPTS cell viability. As shown in Fig. 3, overexpression of E2F1 induced cell death. Cells infected with 100 MOI of GFP-adenovirus for 48 h were used as the control, indicating that death signaling was not induced by adenovirus infection. A background level of cell death, 5.94 ± 1.51%, was detected. Inhibition of Cdk2 by purvalanol in GFP-adenovirus-infected cells lowered the percentage of death cells to 2.07 ± 1.91%. Transduction with 100 MOI of E2F1-adenovirus for 48 h markedly increased cell death to 19.22 ± 2.99% (Fig. 3). Addition of purvalanol (to inhibit Cdk2) together with E2F1-adenovirus reduced the level of cell death to 8.42 ± 2.55%. Cell death in cultured TKPTS cells was also confirmed by cell morphology and nuclear morphological damage (Fig. 4). Overexpression of E2F1 induced cell membrane blebbing and DNA condensation, which were protected via Cdk2 inhibition by purvalanol. Thus, without Cdk2 activity, a high level of cellular E2F1 did not induce death signaling. These results confirmed that cellular accumulation of E2F1 can lead to cell death in TKPTS cells. Furthermore, these results indicated that Cdk2 acted both upstream and downstream of E2F1, possibly through a positive feedback mechanism (see DISCUSSION).


Figure 3
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  		Fig. 3. Overexpression of E2F1 in cultured TKPTS cells induces cell death. Bar graph of trypan blue extrusion assay for cell viability is shown. TKPTS cells treated with 100 MOI green fluorescence protein (GFP)-adenovirus (control), treated with 100 MOI of E2F1-adenovirus (E2F1) for 48 h, treated with E2F1-adenovirus together with 9 µM purvalanol (E2F1+Purvalanol), and GFP-adenovirus together with purvalanol (Purvalanol) were stained with 0.2% trypan blue. Cells unable to extrude trypan blue and thus stained blue were considered nonviable, whereas those expelling dye and unstained were considered viable. The stained and nonstained cells were counted separately on a hemocytometer using an inverted microscope. The number of stained cells, expressed as a percentage of total cells, was used as a measure of cell death. Values are means ± SE from 4 separate cultures.

 

Figure 4
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  		Fig. 4. Light and fluorescence microscopy of cell death induced by E2F1 overexpression in TKPTS cells. TKPTS cells grown on coverslips (scale indicated) were either treated with 100 MOI of E2F1-adenovirus (A and C) for 48 h or treated with E2F1-adenovirus together with 9 µM purvalanol (B and D). TKPTS cells were photographed with an inverted microscope (A and B) using Hoffman optics with a x40 objective before fixation (typical apoptotic cells with membrane blebbing indicated by arrows). Fixed cells were stained with 4',6'-diamidino-2-phenylindole (DAPI), and fluorescence images (C and D) were taken using a fluorescence microscope with a DAPI filter with a x100 oil objective (nuclear condensation indicated by arrows).

 
E2F1 Deficiency Ameliorates Cisplatin Nephrotoxicity

To determine the involvement of E2F1 in cisplatin-induced nephrotoxicity in vivo, we used the E2F1 knockout mouse model. There were no functional or phenotypic differences between untreated E2F1 knockout and wild-type mice (data not show). A single dose of cisplatin (20 mg/kg) was injected into E2F1 knockout and wild-type mice intraperitoneally. Mice were killed 3 days after cisplatin injection, and then kidney function and morphology were assessed. To determine kidney function, BUN and serum creatinine levels were measured using serum that was collected from the retroorbital vein. As shown in Fig. 5, after 3-day cisplatin injection, wild-type mice showed severe kidney failure by both BUN and serum creatinine, which increased up to 189.9 ± 33.6 and 2.34 ± 0.44 mg/dl, respectively. However, E2F1 knockout mice showed significant protection of kidney function: BUN of 92.9 ± 14.2 mg/dl (P = 0.0240 vs. wild-type mice, n = 6/group) and creatinine of 1.33 ± 0.07 mg/dl (P = 0.0457 vs. wild-type mice, n = 6/group). Thus the data indicate that E2F1 deficiency ameliorates cisplatin-induced acute kidney injury.


Figure 5
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  		Fig. 5. Protection of kidney function in E2F1 knockout mice 72 h after cisplatin injection. A: blood urea nitrogen (BUN) levels after cisplatin administration. Values are means ± SE of at least 6 mice and are expressed as mg/dl. In a comparison of statistical differences between cisplatin-injected E2F1 knockout (KO) mice and cisplatin-injected wild-type (WT) mice, *P = 0.02402 at day 3 (*P < 0.05). B: serum creatinine levels after cisplatin administration. Values are means ± SE of at least 6 mice and are expressed as mg/dl. In a comparison of statistical differences between cisplatin-injected E2F1 KO mice and cisplatin-injected WT mice, *P = 0.04570 at day 3 (*P < 0.05).

 
Necrosis also was markedly reduced in E2F1 knockout mice, showing that both kidney function and morphology (Fig. 6) were protected. An assessment of the morphological differences (Fig. 7) demonstrated that parameters of nephrotoxicity, such as necrosis, brush border loss, neutrophil invasion, and tubular dilatation were significantly lower in E2F1 knockout mice. These results suggest that E2F1 is involved in the development of cisplatin nephrotoxicity and cisplatin-induced cell death is at least partly through an E2F1-dependent pathway.


Figure 6
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  		Fig. 6. Morphology of kidney 72 h after cisplatin injection. Representative sections of cisplatin treated WT (A) or E2F1 KO mouse kidney (B) are shown. Scale is indicated.

 

Figure 7
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  		Fig. 7. Morphological evaluation of mouse kidneys 72 h after cisplatin injection expressed on a scale from 0 to 4. Values are means ± SE of kidney sections from at least 6 mice from WT or E2F1 KO mice. Statistically significant differences are indicated: *P = 0.031586; **P = 0.005596; ***P = 0.005727; ****P = 0.002703.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We previously reported that induction of p21 in the kidney after toxic injury caused by cisplatin was a natural, protective mechanism (30) in which the p21 protection is by direct inhibition of Cdk2 activity (38, 51). We recently extended our work by showing that cisplatin cytotoxicity depends on Cdk2, and inhibition of Cdk2 protected kidney cells from cisplatin-induced cell death both in vivo and in vitro (39). These studies provided convincing evidence for the involvement of a key cell cycle regulator in controlling pathways of cell death. To understand the mechanism of cisplatin cytotoxicity, we investigated the pathways and substrates regulated by Cdk2 in response to cisplatin. In the present study, we show that E2F1 was involved in cisplatin-induced cell death and its induction was downstream of Cdk2. A summary of these pathways is presented in Fig. 8.


Figure 8
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  		Fig. 8. Activation of competing death and survival pathways by cisplatin. Exposure of kidney cells in vitro or in vivo activates both cdk2 and p21. The cdk2, in turn, causes E2F1 activation. E2F1 can result in cell death both by transcription-dependent and -independent mechanisms and can also augment cdk2 activation. The p21 protein, by inhibiting cdk2, promotes cell survival. In addition, cell death can result from cdk2-dependent but E2F1-independent mechanisms.

 
In normal cells, cell cycle entry and progression are dependent on Cdk-mediated retinoblastoma (pRb) phosphorylation and subsequent activation of E2F gene family transcription factors, especially E2F1, E2F2, and E2F3 (18, 44). E2F1 is unique among the E2F family members because of its dual function: it triggers both proliferation and cell death (2, 5, 24, 44). E2F2 and E2F3 overlap the role of E2F1 in proliferation. Expression of E2F1, E2F2, and E2F3 can drive cells from quiescence to enter the cell cycle (29, 48), and mouse embryonic fibroblasts (MEFs) derived from E2F1 knockout mice showed a delayed exit from the G0 phase (48). That E2F1 signals apoptosis and functions as a tumor suppressor have been uncovered using E2F1knockout mice, which exhibit defects in apoptosis and have a high incidence of tumor development (8, 50). Moreover, overexpression of E2F1, but not other E2Fs, was shown to induce apoptosis in cultured cells (5, 20, 40) and transgenic mice (14).

E2F1 is a downstream target of pRb and Cdks (17). Association of pRb with E2F1 masks the transcriptional activation domain of E2F1. Importantly, pRb also functions as an active transcriptional repressor by recruiting various cofactors, many of which are involved in remodeling chromatin (45). E2F1-induced apoptosis has been shown to occur by both transcription-dependent and -independent mechanisms (1, 15, 36, 43), and can be inhibited by the expression of pRb (15, 35). The genetic evidence of E2F1 being a critical downstream target for pRb in vivo was provided by two sets of data showing that Rb+/– E2F1–/– mice live longer, that their incidence of pituitary tumors is reduced compared with that of Rb+/– mice (49), and that Rb–/– E2F1–/– embryos survive longer than Rb–/– embryos (47). Furthermore, aberrant apoptosis caused by homozygous pRb deletion in mice was shown to depend on E2F1 (47). pRb serves as a transducer between the cell cycle machinery (cyclin-Cdk complexes) and the E2F family of transcription factors, and its activity is regulated by Cdk phosphorylation. Hyperphosphorylation of pRb mainly by Cdk4 and/or Cdk2 at different sites releases E2F1 to induce gene expression (3, 52). In addition, cyclin A-Cdk2 has been shown to bind and phosphorylate E2F1 to modulate its activity (6, 19, 22). Thus E2F1 activity is tightly controlled by Cdks through pRb, by stable physical interaction, and by direct phosphorylation.

Many lines of evidence have indicated that E2F1 activity affects both cell cycle progression and cell death (2, 5, 44). We now show that cellular E2F1 accumulation correlates with increased Cdk2 activity and cell death in response to cisplatin in cultured TKPTS cells and in mouse kidneys (Fig. 1). Also, this increase of E2F1 protein level was associated with its transcriptional activity (Fig. 1A), although Bell et al. (1) reported that E2F1-directed transcription is not necessary for E2F1-dependent cell death. E2F1 accumulation was sufficient to cause cell death in vitro (Figs. 3 and 4). E2F1 upregulation has been reported to be regulated by transcription (17, 33) and by posttranslational modulation, such as phosphorylation (25). We showed that cisplatin-induced E2F1 accumulation was regulated by Cdk2 and that cisplatin administration failed to induce an increase in E2F1 protein levels in the absence of Cdk2 activity (Fig. 1). Thus E2F1 was identified as a downstream effector of Cdk2 during cisplatin-induced cell death in vitro and in vivo. At the same time, it is also possible that E2F1 acts as an upstream effector of Cdk2 due to a positive feedback loop mechanism. Cdk2 binding partners (cyclin E and cyclin A) as well as E2F1 itself have E2F binding sites within their promoter regions (12, 17, 34). Free E2F1, which binds to these promoter regions, increases the expression of E2F1, cyclin E, and cyclin A, thus regulating positive feedback loops. These may explain why inhibition of Cdk2 activity by purvalanol also protected TKPTS cells from E2F1-induced cell death (Figs. 3 and 4).

We determined that E2F1 functions to induce death signaling during cisplatin cytotoxicity by TopBP1 protein expression, which binds the NH2-terminal region of E2F1, but not other E2Fs, and specifically represses the E2F1-mediated apoptosis pathway (27). As shown in Fig. 2, TopBP1 dramatically protected cultured TKPTS from cisplatin cytotoxicity. Thus, in vitro, cisplatin-induced cell death was through an E2F-mediated pathway.

More importantly, the involvement of E2F1 in cisplatin-induced cell death was confirmed by in vivo experiments. E2F1 knockout mice were more resistant to cisplatin-induced toxic injury by both functional (Fig. 5) and histological (Figs. 6 and 7) criteria demonstrating that E2F1-induced death signaling participated in cisplatin nephrotoxicity. Taken together, we concluded that cisplatin cytotoxicity in vitro and in vivo is substantially mediated through E2F1-dependent pathway(s) downstream of Cdk2.

E2F1 deficiency provided partial protection of kidney function from cisplatin-induced toxic injury in vivo, which was less protective than was afforded by Cdk2-inhibitory drug (39). Although it is possible that partial restoration of E2F1-dependent pathways could arise by functional compensation by other proteins, other explanations for this decreased protection are possible. Several cell death pathways induced by cisplatin in vitro and in vivo have been reported (10, 26, 41), and Cdk2 may be involved in regulating these pathways. Consistent with this, we found that Cdk2-inhibitory drugs protected cultured TKPTS cells from death induced by other intrinsic pathway inducers (Yu F, Megyesi J, and Price PM, unpublished observations). It is likely that cisplatin initiated more than one distinct cell death pathway in which the common element is Cdk2 dependency. One of these pathways acts through both Cdk2 and E2F1, whereas the other pathway(s) is E2F1 independent. Understanding the complexity of death pathways will provide valuable information for controlling cisplatin cytotoxicity and also provide insight into other cell death pathways in general.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases research Grant R01-DK-54471, a VA Merit Review grant, and with resources and use of the facilities at the John L. McClennan Memorial Veterans Affairs Hospital (Little Rock, AR).


   ACKNOWLEDGMENTS
 
We thank Dr. Weei-Chin Lin (University of Alabama at Birmingham) for providing TopBP1 adenovirus and E2F1 adenovirus, Drs. Sander van den Heuvel and Ed Harlow (Massachusetts General Hospital) for clones of human wild-type and dominant-negative Cdk2 cDNA, Dr. Elsa Bello-Reuss (University of Texas Medical Branch) for providing the TKPTS, and Dr. Bert Vogelstein (Johns Hopkins University School of Medicine) for providing the adenoviral construction materials and protocols.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. M. Price, Dept. of Internal Medicine, VA Medical Center, 4300 W. 7th St., Mail Route 151, Little Rock, AR 72205 (e-mail: pricepeterm{at}uams.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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