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Roles of E2F1 in mesangial cell proliferation in vitro
Kidney International, Vol. 56 (1999), pp. 2085–2095
Roles of E2F1 in mesangial cell proliferation in vitro SEIJI INOSHITA, YOSHIO TERADA, OSAMU NAKASHIMA, MICHIO KUWAHARA, SEI SASAKI, and FUMIAKI MARUMO Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Roles of E2F1 in mesangial cell proliferation in vitro. Background. The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. Methods. To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. Results. E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. Conclusions. In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins.
Mesangial cell proliferation is an important feature of mesangial proliferative glomerulonephritis, which progresses to end-stage renal disease [1, 2]. Although a variety of therapies have been used to retard the progression of renal impairment, no curative treatment has yet been devised to suppress mesangial cell proliferation [3, 4]. To design a rational therapy, we must first elucidate the regulation of mesangial cell proliferation. The recent Key words: glomerulonephritis, cell cycle, G1/S cell phase, transcription factor, apoptosis. Received for publication November 17, 1998 and in revised form July 13, 1999 Accepted for publication August 3, 1999
1999 by the International Society of Nephrology
advances in the cell cycle regulation have been accompanied by advances in our understanding of the mechanisms that regulate mesangial cell proliferation. Certain pathogenic mitogens increase the expression of G1 cyclins, cyclin D1, cyclin E, and cyclin A, in mesangial cells in vitro [5–9]. Shankland et al have also demonstrated an increased cyclin D1 and cyclin A expression in experimental glomerulonephritis in vivo [10]. The conjunction of these G1 cyclins with cyclin-dependent protein kinases (CDKs) facilitates phosphorylation of retinoblastoma protein (pRb) families with the release of the E2F family [11, 12]. The E2F family is identified as five closely related proteins (E2F1 through E2F5) [13–16]. The E2F transcription factor regulates cell cycle progression by influencing the expression of proteins required for the G1/S phase transition and DNA synthesis [17]. Although the E2F family is considered critical for the passage of cells through the G1 and into the S phase [18, 19], the role of the E2F family in the mesangial cell cycle has not been clearly elucidated. Several recent studies have showed that E2F decoy oligonucleotides could suppress mesangial cell proliferation in vitro [20] and in vivo [21]. These findings suggest that suppression of E2F transcriptional activity could lead to a novel therapeutic approach. However, the role of the E2F family has not been directly shown in mesangial cells. E2F and G1 cyclins are known to form a complex network and to cooperate in promoting cell cycle progression, but these interactions have not been examined in mesangial cells. G1 cyclins and E2F itself have E2F binding sites within their promoter regions [22–26]. Free E2F, which binds to these promoter regions, increases the expression of E2F [23], cyclin E [24], and cyclin A [26], and regulates positive and negative feedback loops in certain cell lines. The mesangial cells are characterized by a fully differentiated morphology and rarely proliferate in adult animals [27]. The expressions of cyclin D1, cyclin E, and cyclin A are rarely detected in normal quiescent glomerulus [28]. Investigation of the interactions between cyclins and E2F will broaden our understanding of mesangial cell cycle control and help us to decide therapeutic means to suppress mesangial cell proliferation.
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In this study, we showed E2F function directly by using adenovirus-mediated overexpression in primary cultured cells, to our knowledge, for the first time. E2F1 overexpression promoted mesangial proliferation and increased the expressions of cyclins D1, E, and A. Our data suggest that E2F1 plays a central role in the mesangial cell cycle. METHODS Adenoviruses Replication-defective, recombinant adenovirus expressing E2F1 (AdE2F1), driven by the cytomegalovirus (CMV) immediate-early promoter, was kindly provided by Dr. J.R. Nevins [29]. A recombinant adenovirus expressing the LacZ gene (AxCALacZ) was kindly provided by Dr. I. Saito [30]. Each adenovirus was propagated, purified, and titrated by plaque assay on 293 cells. Viral stocks were stored at 2808C and thawed on ice for five minutes before use. Mesangial cell culture and adenovirus infection in vitro Mesangial cell strains from male Sprague-Dawley rats were isolated and characterized as previously reported [31]. Cells were maintained in RPMI1640 medium supplemented with 20% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenite, 10 mm nonessential amino acids, and 10 mm sodium pyruvate at 378C in a 5% CO2 incubator. We used starved medium containing 100 units/ml penicillin, 100 mg/ml streptomycin, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenite, 10 mm nonessential amino acids, and 10 mm sodium pyruvate in RPMI 1640 medium to make the cells quiescent. The cells used in experiments were from passages 5 to 20. The cells were subcultured in 10 cm dishes or 24-well dishes at a density of 5 3 104 cells/ml, were incubated in medium plus 20% fetal calf serum (FCS) until approximately 80% confluence, and were then incubated in starved medium for 48 hours to make the cells quiescent. When using the adenovirus, the medium was removed, and the viral solution was added to the culture dishes for one hour with shaking every 15 minutes. Subsequently, the cells were incubated in starved medium for the indicated times. Survival cell count To count the number of living cells, we used the Cell Counting Kit-8 (Wakojyunyaku, Osaka, Japan). The mesangial cells were subcultured in 24-well dishes and incubated in starved medium for 48 hours. The cells were stimulated with 20% FCS or infected with adenoviruses in 400 ml medium. After the indicated hours, 40 ml WST-8 (tetrazolium monosodium salt) was added and incubated for two hours at 378C CO2 incubator. We used 100 ml
of supernatant to measure the absorbance of a 450 nm wavelength. [3H]-thymidine incorporation Mesangial cells were plated in 24-well plates. After growing to 80% confluence, they were incubated in starved medium for 48 hours and were transfected with AdE2F1 for 24 hours in starved medium. For the last four hours, the cells were pulsed with 1 mCi [3H]-thymidine (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). After washing the cells three times in ice-cold phosphate-buffered saline (PBS), they were precipitated two times with 10% trichloroacetic acid (TCA), redissolved in 0.5 m NaOH with 0.1% Triton X-100, and counted in Aquasol-2 scintillation cocktail (NEN Research Products, Boston, MA, USA). Preparation of nuclear protein from mesangial cells and Western blot analysis The cells harvested with trypsin were washed in PBS. After centrifugation at 1000 r.p.m. for five minutes at 48C, the pellets were resuspended in buffer containing 10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm ethylenediaminetetraacetic acid (EDTA), 0.1 mm egtazic acid (EGTA), 1 mm dithiothreitol (DTT), and 0.5 mm phenylmethylsulfonyl fluoride (PMSF). Next, after incubation for 15 minutes at 48C, 25 ml of 10% Nonidet P-40 was added, and the extract was centrifuged at 12,000 r.p.m. for 30 seconds at 48C. The nuclei were extracted with homogenization buffer containing 20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm PMSF, 1 mg/ml aprotinin, and 1 mg/ml pepstatin, and incubated for 15 minutes on a shaking platform at 48C. After centrifugation of the nuclear extract at 10,000 r.p.m. for 10 minutes at 48C, the supernatant fraction was stored in aliquots at 2808C. Protein concentrations were determined by the Bradford method using bovine serum albumin (BSA) as a standard. The same amount of nuclear protein was resolved in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to an Immobilon P membrane (Daiichikagaku, Tokyo, Japan). To detect E2F1, E2F2, E2F3, E2F4, cyclin D1, cyclin E, and cyclin A (all antibodies purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), the membrane was incubated with antibody at a dilution of 1:1000 to 1:3000 in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skim milk for three hours at room temperature. After the membrane was washed in TBS containing 0.1% Tween 20, it was incubated with a second antibody in TBS containing 0.1% Tween 20 and 5% skim milk for one hour at room temperature. The membrane was visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech).
Inoshita et al: Mesangial cell cycle regulation by E2F1
Northern blot analysis Total RNA was extracted from the mesangial cells using TRIREAGENTe (Life Technologies, Gaithersburg, MD, USA). Twenty micrograms of each total RNA sample were separated on 1% agarose-formaldehydemorpholino propanesulfonic acid (MOPS) gel and transferred to Hybond-N hybridization membrane (Amersham Pharmacia Biotech). Northern hybridizations were carried out at room temperature for 16 hours in a solution containing 5 3 Denhalt’s solution, 2 3 standard saline citrate (SSC), 0.1% SDS, 100 mg/ml denatured salmon sperm DNA, and 10% dextran sulfate. Full-length 1.5 kb human E2F1 cDNAs were labeled by the random priming method and used as probes for Northern hybridization. Following hybridization, the membranes were washed twice for five minutes at room temperature in 5 3 SSPE and 0.5% SDS, twice at 378C for 15 minutes in 1 3 SSPE and 0.5% SDS, and once at 378C for 15 minutes in 0.1 3 SSPE and 0.5% SDS and were then exposed to Fuji x-ray film (Fuji Photo Film, Kanagawa, Japan) for 24 to 48 hours at room temperature. The films were analyzed by BASe (Fuji Photo). Cell cycle analysis by fluorescence-activated cell sorter (FACS) Mesangial cells were cultured in 10 cm dishes. The cells were incubated in starved medium for 48 hours before adenovirus infection. After removal of the medium, 109 pfu/ml AdE2F1 solutions were added to the culture dishes for one hour with shaking every 15 minutes, and the cells were then incubated in 0.25% FCS medium for the indicated times. The samples were washed twice with PBS and resuspended in 70% ethanol for at least 12 hours at 48C, and then the fixed and permeabilized cells were collected by centrifugation and were washed with PBS. The cells were denatured by RNase, stained with propidium iodide, and analyzed by flow cytometry using a FACS Calibur (Becton Dickinson, San Jose, CA, USA). The percentages of the cells in G1, S, and G2/M phases were then determined. Measurement of caspase 3 activities A Caspase 3 Fluorometric Protease Assay Kit (MBL, Tokyo, Japan) was used for measurement of caspase 3 activities. In brief, cells were plated in six-well dishes, cultured in starved medium for 48 hours, transfected by adenovirus, and incubated in starved medium that was changed once daily. At indicated times, the cells were collected and lyzed in lysis buffer, and the protein concentration was normalized by the Bradford assay. The lysates were incubated with the same amount of reaction buffer and 50 mm DEVD-AFC substrate for two hours at 378C. Fluorescence was monitored with an excitation wavelength of 400 nm and an emission wavelength of 505 nm.
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Transient transfections and luciferase assays Transfection assays were performed in rat mesangial cells by electroporation methods. Mesangial cells were grown in 20% FCS containing medium until 80% confluence and were then incubated in starved medium for 48 hours to stop the cell cycle. After stimulating the cells with FCS-containing medium for 24 hours, they were washed by PBS, collected by trypsinization, and centrifuged and washed twice by PBS. The cells were put in 0.4 cm cuvettes with 5 mg luciferase reporter plasmid and 5 mg b-galactociderse reporter plasmid, and then they were transfected by electroporation at 300 V using a Bio-Rad gene pulser (Bio-Rad, Hercules, CA, USA). Luciferase reporter plasmids driven by the human cyclin D1 promoter were provided by Dr. M. Eilers. The human cyclin E promoter was provided by Dr. K. Ohtani, and the human cyclin A promoter was provided by Dr. J. Sobczak-Thepot. The cells were incubated in FCS-containing medium for 24 hours and infected by adenovirus. Luciferase activity was measured as described previously 12 hours after infection. CDK4 and CDK2 kinase assay The immune complex kinase assay was performed using essentially the same methods described by Matsushime et al [32]. The mesangial cells were incubated with serumstarved medium for 48 hours. The cells were infected with 109 pfu/ml AdE2F1 or were stimulated by 20% FCS as a positive control. For CDK4 and CDK2 kinase assays, the lysates were prepared 8 and 12 hours after infection, respectively. Immune complexes were recovered by centrifugation after incubating the cell lysates for two hours at 48C with 10 ml of anti-cyclin D1 antibody or anti-cyclin E antibody (Santa Cruz Biotechnology Inc.) and then one hour with 30 ml of protein G-plus agarose (Oncogene Science, Uniondale, NY, USA). Immunoprecipitated proteins were suspended in 30 ml of kinase buffer [50 mm HEPES (pH 7.5), 10 mm MgCl2, 1 mm DTT] containing substrate [0.2 mg of soluble glutathione S-transferasepRb fusion protein (Santa Cruz Biotechnology Inc.) for CDK4 kinase assay, and Histione H1 (Boeringer Mannheim, Germany) for CDK2 assay] and 2.5 mm EGTA, 10 mm b-glycerophosphate, 1 mm NaF, 20 mm ATP, and 10 mCi of [g-32P] ATP (NEN, DuPont, Boston, MA, USA). After incubation for 30 minutes at 308C with occasional mixing, the samples were boiled in polyacrylamide gel sample buffer containing sodium dodecyl sulfate and separated by electrophoresis. Phosphorylated proteins were analyzed by BAS station. Statistical analysis The results were given as means 6 sem. The significance of difference was tested using analysis of variance in multiple comparisons. P , 0.05 was considered statistically significant.
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Fig. 1. (A) Increase of the number of mesangial cells after fetal calf serum (FCS) stimulation. The mesangial cells were incubated in starved medium for 48 hours and were then stimulated by replacement with 20% serum-containing medium. After indicated times, the cells were incubated with WST-8 for two hours. Absorbance that reflected the number of living cells was measured. Each point represents the mean 6 sem of four experiments. *P , 0.05 vs. control; **P , 0.05 vs. 18 hours by analysis of variance (ANOVA) test. (B) Time course of [3H]-thymidine incorporation after serum stimulation. The mesangial cells were treated as described in (A). After indicated times, the cells were incubated with [3H]-thymidine for two hours. Each bar represents the mean 6 sem of four experiments. *P , 0.05 vs. control by ANOVA.
RESULTS Time course of the mesangial cell cycle To characterize mesangial cell proliferation, we examined cell number and DNA synthesis after growth stimulation. Mesangial cells were made quiescent by incubation in starved medium for 48 hours and then stimulated by replacement in medium containing 20% FCS. After the indicated times, the cells were incubated with WST-8 for two hours, and then the absorbance that reflects the surviving cell number was measured. Figure 1A shows that the number of mesangial cells increased by about twofold at 18 hours. This data indicated that the M phase started between 12 and 18 hours after FCS stimulation in our condition. We examined [3H]-thymidine incorporation after FCS stimulation (Fig. 1B). The mesangial cells were treated as described earlier in this article and then incubated with [3H]-thymidine for two hours. [3H]thymidine incorporation was significantly increased 6 hours after FCS stimulation and reached at its peak after 12 hours. These data suggested that almost all of the mesangial cells entered the S phase at some point between 6 and 12 hours and exited the S phase at some point between 12 and 18 hours. To be brief, the majority
of mesangial cells pass through the G1/S phase at some point between 6 and 12 hours and then pass through the G2/M phase at some point between 12 and 18 hours. Expression of the E2F family in rat mesangial cells To determine which member of the E2F family is important and involved in mesangial cell proliferation, the time course of protein expression of the E2F family after FCS stimulation was examined, and it was compared with the mesangial cell cycle discussed earlier in this article. Mesangial cells were rendered quiescent and stimulated by FCS using the same methods described in Figure 1. Nuclear protein was extracted at indicated times. As shown in Figure 2A, protein expression of E2F1 through E2F3 was induced by FCS stimulation. The expression of E2F1 was almost undetectable in a quiescent state and was only slightly detectable at six hours after FCS stimulation. It strongly increased from 12 hours after FCS stimulation. This time course was consistent with the result of [3H]-thymidine incorporation. Although protein expression of E2F2 and E2F3 resembled that of E2F1, magnitudes of their changes were notably smaller than that of E2F1 expression. The increases of E2F2 and E2F3 were obscure compared
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Fig. 3. Time course of E2F1 overexpression after AdE2F1 infection. The mesangial cells were incubated in starved medium for 48 hours and infected with 109 pfu/ml AdE2F1. Nuclear protein was extracted after indicated times. Western blotting was performed with same amount of nuclear protein.
Fig. 2. (A) Induction of E2F family (E2F1 through E2F4) protein expression after serum stimulation in rat mesangial cells. Western blot analysis was performed with a specific antibody for E2F family individuals. Primary cultured rat mesangial cells were incubated in starved medium for 48 hours to render them quiescent. Nuclear protein was extracted at indicated times after stimulation with 20% FCS. Aliquots of the nuclear protein were electrophoresed on a SDS-polyacrylamide gel and visualized by the ECL chemiluminescence system. (B) Induction of E2F1 mRNA expression after stimulation in rat mesangial cells. The mesangial cells were incubated in starved medium for 48 hours to render them quiescent. Total RNA was extracted at indicated times after stimulation with 20% FCS extracted as described in the Methods section. Aliquots of total RNA (20 mg per lane) were electrophoresed on a 1% agarose gel. The membrane was hybridized sequentially using a specific cDNA probe for E2F1. Ribosomal RNA (18S) is shown as a control for RNA loading.
with the increase of E2F1 in the G1/S phase transition. In contrast, protein expression of E2F4 was highest among the E2F family in the quiescent state, but it showed only a slight increase during FCS stimulation. The expression of E2F4 increased at six hours. E2F4 appeared to be mainly expressed in cytosols without growth simulation [33, 34]. When we extracted nuclear protein to detect slight changes after growth stimulation, it was expected that E2F4 expression at zero hours, including cytosolic fraction, would exceed the expression we detected here. Some studies showed E2F4 did not change the amount of expression through the cell cycle and had no influence in promoting the cell cycle [35]. These data suggest that the E2F1, E2F2, and E2F3 correlate with mesangial cell proliferation. The peak of expression of E2F3 at 18 hours was late for a peak in DNA synthesis. In fact, which protein, E2F1 or E2F2, plays a more important role in the mesangial cell cycle could
not be distinguished. We focused on E2F1 in the following study because the E2F1 expression exceeded E2F2 and E2F3 expression during cell cycle progression in mesangial cells. The time course of the expression of E2F1 mRNA was also examined. Figure 2B shows that E2F1 mRNA increased moderately at three hours after FCS stimulation and increased strongly at 12 hours. The time course of mRNA expression was almost the same as that of protein expression. The similarity between these time courses suggested that the transcriptional regulation of E2F1 is important in the mesangial cell cycle. Regulation of cell cycle by E2F1 overexpression in rat mesangial cells Adenovirus-mediated gene transfer was used to investigate whether the overexpression of E2F1 could promote cell cycle progression. In our previous study, we demonstrated the efficiency of transfection and the toxicity by means of adenovirus-mediated transfection to mesangial cells [6]. At first the time course of E2F1 protein expression was examined after AdE2F1 infection. The quiescent mesangial cells were infected with 109 pfu/ml AdE2F1. Then the mesangial cells were harvested at indicated times and the nuclear protein extracted. Figure 3 shows that E2F1 expression significantly increased 6 hours after infection and continued thereafter up to at least 30 hours. We investigated whether only E2F1 overexpression could promote DNA synthesis and cell cycle progression. The quiescent mesangial cells were infected by various concentrations of AdE2F1 for 24 hours, and then the [3H]-thymidine incorporation was measured. As shown in Figure 4, overexpression of E2F1 in rat mesangial cells increased thymidine uptake to 220% in the absence of FCS stimulation, which is an almost equal level to that induced by 20% FCS stimulation. Next, the mesangial cell cycle progression was examined using flow cytometry. Mesangial cells were infected with 109 pfu/ml AdE2F1, after indicated times harvested,
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Fig. 4. Effect of E2F1 overexpression on mesangial cell proliferation as assessed by [3H]-thymidine incorporation. Quiescent mesangial cells cultured in starved medium for 48 hours were incubated with indicated dosages of AdE2F1. Control mesangial cells were infected with 109 pfu/ml AdLacZ. Twenty-four hours after incubation, they were pulsed with [3H]thymidine for four hours. Each bar represents the mean 6 sem of four experiments. *P , 0.05 vs. control by analysis of variance test.
and were investigated to determine the amount of DNA content. Figure 5A illustrates an experimental plotting of the cell number as a function of DNA content at 0, 12, and 24 hours. The data, summarized in Figure 5B, revealed that overexpression of E2F1 alone significantly increased the percentage of the S phase and decreased the percentage of the G0/G1 phase. The percentage of the G2/M phase was increased by E2F1 overexpression, although it is not statistically significant. These data suggest that mesangial cells progress from the G1 phase to the S phase at same point from six hours after E2F1 overexpression and then onward to the M phase at same point from 18 hours. We concluded that overexpression of E2F1 alone could accelerate the G1/S phase transition and promote mesangial cell proliferation. In other words, E2F1 upregulation is sufficient for rat mesangial cell proliferation in certain conditions. Induction of apoptosis by E2F1 overexpression in rat mesangial cells Some studies reported that overexpression of E2F1 induced apoptosis after the induction of the S phase
Fig. 5. Cell cycle analysis using flow cytometry after E2F1 overexpression. Mesangial cells were brought to quiescence as described in Figure 3, were infected with 109 pfu/ml AdE2F1, and subsequently collected at indicated times. The cells were stained with propidium iodine and processed for flow cytometry. (A) Representative tracing. The horizontal axis reflects relative DNA content and the vertical axis represents cell number. (B) The percentage of gated control and AdE2F1-infected cells in each phase of the cell cycle was graphed from the flow cytometric analysis. Each bar represents the mean 6 sem of four experiments. *P , 0.05 vs. 0 hours by ANOVA.
[36–39]. We observed the cell viability over a long time course after the overexpression of E2F1. Mesangial cells were cultured in starved medium to maintain quiescence for two days. Next, we transfected cells with 109 pfu/ml AdE2F1 or with the same amount of AxCALacZ as a control. After the indicated times, we incubated the cells with WST-8 for two hours and then measured the absorbance that reflects the surviving cell number. Figure 6A showed a twofold increase in the number of living mesangial cells 24 hours after E2F1 overexpression. Thereafter, the number of living mesangial cells quickly decreased from 48 hours, whereas the number of control cells gradually increased up to 72 hours. Caspase 3 protease activity was measured to determine whether this decrease from 48 hours reflected apoptosis. The mesangial cells were treated in the same manner described in Figure 6A. After the indicated times, cell lysates were prepared and normalized by protein concentration.
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Figure 6A. These data suggested that E2F1 overexpression completed the mesangial cell cycle and doubled the number of living cells. After that, E2F1 overexpression induced apoptosis.
Fig. 6. (A) Change the number of living mesangial cells after E2F1 overexpression. The mesangial cells were incubated for 48 hours in starved medium. After that, the cells were infected with 109 pfu/ml AdE2F1 (———) or AxCALacZ (- - - - -) as a control. The starved medium was changed every day. After the indicated times, the cells were incubated with WST-8 for two hours. The absorbance that reflects the number of surviving cells was measured. Each point represents the mean 6 sem of three experiments. (B) Induction of caspase 3-like protease activity by E2F1 overexpression. The mesangial cells were treated as described in (A) and were infected with 109 pfu/ml AdE2F1 (j) or AxCALacZ ( ) as a control. After indicated times, cell lysates were prepared and normalized by protein concentration. Caspase 3-like protease activity was measured by proteolytic cleavage of the specific fluorogenic substrate DEVD-AFC. Each bar represents the mean 6 sem of three experiments.
Caspase 3-like protease activity was measured by proteolytic cleavage of the specific fluorogenic substrate DEVD-AFC. Caspase 3-like protease activity was significantly increased at 48 and 72 hours compared with control, but it was lower than control at 24 hours. This increase was consistent with the time course showed in
Induction of the G1 cyclins, cyclin D1, cyclin E, and cyclin A by E2F1 overexpression To elucidate the mechanisms of E2F1-induced mesangial cell cycle progression, we investigated the interaction between E2F1 and G1 cyclins. We previously reported that G1 cyclins play an important role in the mesangial cell cycle [6, 9]. These G1 cyclins are well known to have E2F binding sites in their promoter region [22, 24–26]. However, the network of G1 cyclins and E2F1 has not been clear, and the interaction between G1 cyclins and E2F has not been investigated in primary cultured mesangial cells. To clear the regulation of G1 cyclins and E2F1 in detail, we studied the promoter activity and protein expression of G1 cyclins after E2F1 overexpression. We transfected plasmids containing the cyclin D1, cyclin E, or cyclin A promoter region with luciferase reporter gene to rat mesangial cells by electroporation methods, and then E2F1 was overexpressed using adenovirus vector. Figure 7A showed that E2F1 overexpression significantly increased cyclin D1, cyclin E, and cyclin A promoter activity 5.7-fold, 30.8-fold, and 12.8-fold, respectively. Figure 7B showed the time course of G1 cyclin protein expressions after AdE2F1 infection. The quiescent mesangial cells were infected with AdE2F1 in starved medium. Surprisingly, overexpression of E2F1 caused a sequential induction of three G1 cyclins identical to a sequential induction triggered by growth stimulation. E2F1 increased cyclin D1 from 6 hours up to a peak at 12 hours, and it increased cyclin E and cyclin A from 12 and 18 hours, respectively. We also performed quantitative analysis of protein expressions of G1 cyclins (Fig. 7C). This result was consistent with the time course of flow cytometry analysis. The mesangial cells entered the S phase from 6 hours after E2F1 overexpression and exited the S phase from 18 hours. We examined whether this induction of G1 cyclins contributed to the increased kinase activities of CDK4 and CDK2, respectively. Eight hours of AdE2F1 infection increased CDK4 kinase activity to a level almost equal to that induced by eight hours of FCS stimulation. (Fig. 8, upper panel). CDK2 activity was also increased 2.8 times by E2F1 overexpression for 12 hours (Fig. 8, lower panel), but it was only minimally increased by E2F1 overexpression for eight hours (data not shown). These data suggest that E2F1 initiates G1/S phase transition not only by induction of genes required for DNA synthesis, but also by induction G1 cyclins. E2F1 and G1 cyclins strongly cooperate in promoting the G1/S phase transition in mesangial cells.
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Fig. 7. (A) Effects of E2F1 overexpression on the transcriptional activity of the cyclin D1, cyclin E, and cyclin A promoters. Mesangial cells were equilibrated to the cell cycle phase and subsequently grown in serum containing medium as described in the Methods section. The cells were transfected with cyclin D1, cyclin E, or cyclin A promoter plasmids containing luciferase reporter gene by the electroporation method. After an additional 24 hours of growth, the cells were infected with indicated dosages AdE2F1 or AxCALacZ (control). The cells were collected and assayed for luciferase activities. The results represent the means 6 sem of relative luciferase activities (after collection by protein concentration) from four experiments. (B and C) Effect of E2F1 overexpression on the induction of the cyclin D1 (r), cyclin E (j), and cyclin A (m) proteins. Mesangial cells were brought to quiescence by 48hour serum withdrawal and infected with 109 pfu/ml AdE2F1. Nuclear proteins were extracted indicated times after infection. Aliquots of the nuclear protein were separated on a SDS-PAGE and visualized by the ECL chemiluminescence system (B). Quantitative analysis of protein expressions of G1 cyclins was performed using DNAquant (C).
DISCUSSION In this study, we demonstrated for the first time that E2F expression was a necessary and sufficient condition for mesangial cell proliferation. Moreover, the same case may hold true in many other cell lines as well. The p16/ Rb/E2F regulatory pathway is one of the most frequent targets of genetic alterations in human cancers [40]. This suggests that the regulation of this pathway differs in carcinoma cells and primary culture cells. Several recent studies showed that E2F and G1 cyclins were effective therapeutic targets of mesangial proliferative disease [20, 21, 41]. We therefore thought it was important to clarify the regulation of mesangial cell cycle by E2F for future therapeutic use. We showed that E2F1, E2F2, and E2F3 expressions were increased by serum stimulation, whereas E2F4 expression was not (Fig. 2A). E2F1, E2F2, and E2F3 are well known to be distinctly different from E2F4. Namely, the former stimulate cell cycle progression, whereas the latter does not [42]. However, there are no clear differences between E2F1, E2F2, and E2F3 in terms of their ability to induce cell cycle progression. In mesangial cells,
the peak in E2F3 expression was late for S phase, and E2F1 and E2F2 expressions increased over a time course consistent with that of G1/S phase transition. E2F1 expression increased 20-fold from 6 to 12 hours, whereas E2F2 expression increased by no more than twofold. We presumed that E2F1 was a major protein among the E2F family in mesangial cell cycle progression. E2F1 overexpression increased [3H]-thymidine incorporation and the percentage of cells in the S phase. In our flow cytometry study, E2F1 overexpression tended to increase the percentage of cells in the G2/M and G0/G1 phases at 24 hours, with a twofold increase in the number of living cells at that time point. The percentage of cells that progressed to S and G2/M phases was smaller than the percentages previously reported in other cell lines. We recognize that this discrepancy may be due to the tendency of mesangial cells to stay quiescent, the high expression of cyclin-dependent kinase inhibitor [28], or the efficiency of the gene transfer to the primary culture cell in our system. In any case, these data suggested that E2F1 overexpression could induce the G1/S phase transition and complete mitosis. Mesangial cells have been
Inoshita et al: Mesangial cell cycle regulation by E2F1
Fig. 8. Induction of CDK4 and CDK2 kinase activity by E2F1 overexpression. The mesangial cells were incubated with starved medium for 48 hours and then either infected with 109 pfu/ml AdE2F1 or stimulated by FCS as a positive control. After 8 hours for the CDK4 kinase assay and 12 hours for the CDK2 kinase assay, cell lysates were immunoprecipitated with anti-cyclin D1 antibody and anti-cyclin E antibody, respectively. Kinase assays were performed with Rb protein or Histone H1 as a substrate for CDK4 and CDK2, respectively.
rarely investigated to determine whether the introduction of an exogenous gene facilitates the cell cycle. To our knowledge, this is the first report in which the expression of a single gene promotes cell cycle re-entry and completes mitosis in mesangial cells. It is well known that E2F1 overexpression causes apoptosis [36–39]. The mechanisms of E2F1-induced apoptosis have been reported to act in both p53-dependent and p53-independent manners. Although it remains unclear precisely how E2F1 induces apoptosis, the process is not simply an artifact of protein overexpression. The induction of apoptosis is specific to E2F1 among the E2F family, and neither E2F2 nor E2F3 have been showed to have any connection with apoptosis [43]. In mesangial cells, E2F1 overexpression induced apoptosis at 48 hours after infection. At this time point, mitosis was completed once. Caspase 3 activity was also increased at 48 hours. We speculate that E2F1 overexpression induces proper cell division, whereas continuous E2F1 expression or E2F1-induced gene expression at an inappropriate phase causes apoptosis. Some studies reported that apoptosis was increased in the progression of glomerular sclerosis. Further studies are needed to elucidate the relationship between progression of glomerular sclerosis and E2F1induced apoptosis. Various experiments suggest that free E2F increases the expression of genes required for DNA synthesis, as well as regulatory genes such as c-myc, N-myc, erb-B, c-myb, and B-myb [44–50]. It is also well known that cyclin D1, cyclin E, and cyclin A have E2F binding sites in their promoter regions [22, 24–26]. Although it is established that free E2F induces the expression of cyclin E and cyclin A [24, 26], E2F has not been reported to
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increase the protein level of cyclin D1. Watanabe et al reported that E2F1 overexpression decreased cyclin D1 expression dependent on the Sp1 binding site [51]. Furthermore, Ohtani, Degregori, and Nevins showed that E2F1 overexpression activated cyclin D1 promoter without increasing the protein level [24]. Fan and Bertino suggested that there may be a positive feedback regulation loop between E2F and cyclin D1 by means of a dominant negative mutant of E2F1 [52]. In our data, E2F1 overexpression increased both the promoter activity and protein expression of cyclin D1. Our study is the first to report an interaction between E2F and G1 cyclins in primary cultured cells, and we postulate that the difference in cyclin D1 regulation induced by E2F1 reflects a difference in the mechanisms that regulate G1/S phase transition between primary cultured cells and immortal cells. In our preliminary experiments, E2F1 overexpression increased cyclin D1 promoter activity but not protein expression in LLC-PK1 cells (data not shown). Cyclin D1/CDK4 and cyclin E/CDK2 complexes facilitate phosphorylation of the pRb family with the release of free E2F, and then the released E2F increases G1 cyclins and promotes G1/S phase progression. E2F and G1 cyclins form the firm, positive feedback loop. Late induction of cyclin A may contribute to the reduction of DNA-binding activities of the E2F1-DP-1 dimer [53–55], in other words, form negative feedback loop. Various studies have demonstrated that the expressions of specific cyclins and CDKs and CDK inhibitors change during mesangial cell proliferation in cell culture systems [5–9] and in in vivo experimental models [10]. Some studies indicated that inhibition of CDKs or E2F suppressed mesangial cell proliferation [20, 21, 41]. Smooth muscle cell proliferation after endothelium injury was suppressed by infusion of an E2F decoy oligonucleotide [56]. These experiments suggest new therapeutic approaches to benign proliferative disease. Because most of our knowledge about cell cycle regulation is based on experiments using cells derived from carcinoma, it is important to clarify the difference in the cell cycle regulation between primary culture cells and cell lines. Studies of cell cycle regulatory proteins, including cyclins and E2F in human renal disease, have not yet been reported. Mesangial cell proliferation continues for a few decades in human chronic glomerulonephritis, and it is not clear whether cell cycle regulatory genes such as cyclins and E2F are involved in the pathogenesis of this disease. Further studies are needed to determine whether these cell cycle control mechanisms are involved in such a long-term proliferation. ACKNOWLEDGMENTS We thank for Dr. I. Saito for kindly providing AxCALacZ, and Drs. J.R. Nevins and K. Ohtani for kindly providing AdE2F1 and cyclin E promoter. We are also grateful to Drs. M. Eilers and J.
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Sobczak-Thepot for kindly providing cyclin D1 and cyclin A promoter, respectively. Reprint requests to Yoshio Terada, M.D., Second Department of Internal Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: ATP, adenosine 59-triphosphate; AdE2F1, replication-defective recombinant adenovirus expressing E2F1; AxCALacZ, recombinant adenovirus expressing the LacZ gene; BSA, bovine serum albumin; CDK, cyclin-dependent protein kinase; CMV, cytomegalovirus; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetatic acid; EGTA, egtazic acid; FCS, fetal calf serum; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; WST-8, tetrazolium monosodium salt.
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Roles of E2F1 in mesangial cell proliferation in vitro SEIJI INOSHITA, YOSHIO TERADA, OSAMU NAKASHIMA, MICHIO KUWAHARA, SEI SASAKI, and FUMIAKI MARUMO Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Roles of E2F1 in mesangial cell proliferation in vitro. Background. The proliferation of mesangial cells is a common feature of many glomerular diseases. E2F transcription factors play an important role in the regulation of the cell cycle. However, the regulation of the mesangial cell cycle and the participation of the E2F family (E2F1 through E2F5) in mesangial cells have not been clarified. Therefore, we investigated the roles of the E2F family in the mesangial cell cycle. Methods. To elucidate the importance of the E2F family, we investigated the mesangial cell cycle by examining the cell count and thymidine incorporation, and compared it with the protein expression of E2F. Using adenovirus-mediated gene transfer, the cell cycle and apoptosis were examined by measurement of thymidine incorporation, flow cytometry, and caspase 3 activity. We also studied the interaction between E2F1 and G1 cyclins by promoter assay, Western blotting, and CDK kinase assay. Results. E2F1 increased 20-fold in G1/S phase transition. E2F1 overexpression facilitated the mesangial cell cycle and later induced apoptosis. Furthermore, E2F1 overexpression increased the promoter activities and protein expressions of G1 cyclins, cyclin D1, cyclin E, cyclin A. The up-regulation of G1 cyclins contributed to the activation of CDK4 and CDK2. Conclusions. In mesangial cells, we conclude that E2F1 plays an important role in G1/S phase transition and in apoptosis. E2F1 regulates the mesangial cell cycle through two distinct pathways. First, E2F1 directly transcribes genes that are necessary for DNA synthesis, and second, it promotes cell cycle progression via the induction of G1 cyclins.
Mesangial cell proliferation is an important feature of mesangial proliferative glomerulonephritis, which progresses to end-stage renal disease [1, 2]. Although a variety of therapies have been used to retard the progression of renal impairment, no curative treatment has yet been devised to suppress mesangial cell proliferation [3, 4]. To design a rational therapy, we must first elucidate the regulation of mesangial cell proliferation. The recent Key words: glomerulonephritis, cell cycle, G1/S cell phase, transcription factor, apoptosis. Received for publication November 17, 1998 and in revised form July 13, 1999 Accepted for publication August 3, 1999
1999 by the International Society of Nephrology
advances in the cell cycle regulation have been accompanied by advances in our understanding of the mechanisms that regulate mesangial cell proliferation. Certain pathogenic mitogens increase the expression of G1 cyclins, cyclin D1, cyclin E, and cyclin A, in mesangial cells in vitro [5–9]. Shankland et al have also demonstrated an increased cyclin D1 and cyclin A expression in experimental glomerulonephritis in vivo [10]. The conjunction of these G1 cyclins with cyclin-dependent protein kinases (CDKs) facilitates phosphorylation of retinoblastoma protein (pRb) families with the release of the E2F family [11, 12]. The E2F family is identified as five closely related proteins (E2F1 through E2F5) [13–16]. The E2F transcription factor regulates cell cycle progression by influencing the expression of proteins required for the G1/S phase transition and DNA synthesis [17]. Although the E2F family is considered critical for the passage of cells through the G1 and into the S phase [18, 19], the role of the E2F family in the mesangial cell cycle has not been clearly elucidated. Several recent studies have showed that E2F decoy oligonucleotides could suppress mesangial cell proliferation in vitro [20] and in vivo [21]. These findings suggest that suppression of E2F transcriptional activity could lead to a novel therapeutic approach. However, the role of the E2F family has not been directly shown in mesangial cells. E2F and G1 cyclins are known to form a complex network and to cooperate in promoting cell cycle progression, but these interactions have not been examined in mesangial cells. G1 cyclins and E2F itself have E2F binding sites within their promoter regions [22–26]. Free E2F, which binds to these promoter regions, increases the expression of E2F [23], cyclin E [24], and cyclin A [26], and regulates positive and negative feedback loops in certain cell lines. The mesangial cells are characterized by a fully differentiated morphology and rarely proliferate in adult animals [27]. The expressions of cyclin D1, cyclin E, and cyclin A are rarely detected in normal quiescent glomerulus [28]. Investigation of the interactions between cyclins and E2F will broaden our understanding of mesangial cell cycle control and help us to decide therapeutic means to suppress mesangial cell proliferation.
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In this study, we showed E2F function directly by using adenovirus-mediated overexpression in primary cultured cells, to our knowledge, for the first time. E2F1 overexpression promoted mesangial proliferation and increased the expressions of cyclins D1, E, and A. Our data suggest that E2F1 plays a central role in the mesangial cell cycle. METHODS Adenoviruses Replication-defective, recombinant adenovirus expressing E2F1 (AdE2F1), driven by the cytomegalovirus (CMV) immediate-early promoter, was kindly provided by Dr. J.R. Nevins [29]. A recombinant adenovirus expressing the LacZ gene (AxCALacZ) was kindly provided by Dr. I. Saito [30]. Each adenovirus was propagated, purified, and titrated by plaque assay on 293 cells. Viral stocks were stored at 2808C and thawed on ice for five minutes before use. Mesangial cell culture and adenovirus infection in vitro Mesangial cell strains from male Sprague-Dawley rats were isolated and characterized as previously reported [31]. Cells were maintained in RPMI1640 medium supplemented with 20% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenite, 10 mm nonessential amino acids, and 10 mm sodium pyruvate at 378C in a 5% CO2 incubator. We used starved medium containing 100 units/ml penicillin, 100 mg/ml streptomycin, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml selenite, 10 mm nonessential amino acids, and 10 mm sodium pyruvate in RPMI 1640 medium to make the cells quiescent. The cells used in experiments were from passages 5 to 20. The cells were subcultured in 10 cm dishes or 24-well dishes at a density of 5 3 104 cells/ml, were incubated in medium plus 20% fetal calf serum (FCS) until approximately 80% confluence, and were then incubated in starved medium for 48 hours to make the cells quiescent. When using the adenovirus, the medium was removed, and the viral solution was added to the culture dishes for one hour with shaking every 15 minutes. Subsequently, the cells were incubated in starved medium for the indicated times. Survival cell count To count the number of living cells, we used the Cell Counting Kit-8 (Wakojyunyaku, Osaka, Japan). The mesangial cells were subcultured in 24-well dishes and incubated in starved medium for 48 hours. The cells were stimulated with 20% FCS or infected with adenoviruses in 400 ml medium. After the indicated hours, 40 ml WST-8 (tetrazolium monosodium salt) was added and incubated for two hours at 378C CO2 incubator. We used 100 ml
of supernatant to measure the absorbance of a 450 nm wavelength. [3H]-thymidine incorporation Mesangial cells were plated in 24-well plates. After growing to 80% confluence, they were incubated in starved medium for 48 hours and were transfected with AdE2F1 for 24 hours in starved medium. For the last four hours, the cells were pulsed with 1 mCi [3H]-thymidine (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). After washing the cells three times in ice-cold phosphate-buffered saline (PBS), they were precipitated two times with 10% trichloroacetic acid (TCA), redissolved in 0.5 m NaOH with 0.1% Triton X-100, and counted in Aquasol-2 scintillation cocktail (NEN Research Products, Boston, MA, USA). Preparation of nuclear protein from mesangial cells and Western blot analysis The cells harvested with trypsin were washed in PBS. After centrifugation at 1000 r.p.m. for five minutes at 48C, the pellets were resuspended in buffer containing 10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm ethylenediaminetetraacetic acid (EDTA), 0.1 mm egtazic acid (EGTA), 1 mm dithiothreitol (DTT), and 0.5 mm phenylmethylsulfonyl fluoride (PMSF). Next, after incubation for 15 minutes at 48C, 25 ml of 10% Nonidet P-40 was added, and the extract was centrifuged at 12,000 r.p.m. for 30 seconds at 48C. The nuclei were extracted with homogenization buffer containing 20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm PMSF, 1 mg/ml aprotinin, and 1 mg/ml pepstatin, and incubated for 15 minutes on a shaking platform at 48C. After centrifugation of the nuclear extract at 10,000 r.p.m. for 10 minutes at 48C, the supernatant fraction was stored in aliquots at 2808C. Protein concentrations were determined by the Bradford method using bovine serum albumin (BSA) as a standard. The same amount of nuclear protein was resolved in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred to an Immobilon P membrane (Daiichikagaku, Tokyo, Japan). To detect E2F1, E2F2, E2F3, E2F4, cyclin D1, cyclin E, and cyclin A (all antibodies purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), the membrane was incubated with antibody at a dilution of 1:1000 to 1:3000 in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% skim milk for three hours at room temperature. After the membrane was washed in TBS containing 0.1% Tween 20, it was incubated with a second antibody in TBS containing 0.1% Tween 20 and 5% skim milk for one hour at room temperature. The membrane was visualized using the ECL chemiluminescence system (Amersham Pharmacia Biotech).
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Northern blot analysis Total RNA was extracted from the mesangial cells using TRIREAGENTe (Life Technologies, Gaithersburg, MD, USA). Twenty micrograms of each total RNA sample were separated on 1% agarose-formaldehydemorpholino propanesulfonic acid (MOPS) gel and transferred to Hybond-N hybridization membrane (Amersham Pharmacia Biotech). Northern hybridizations were carried out at room temperature for 16 hours in a solution containing 5 3 Denhalt’s solution, 2 3 standard saline citrate (SSC), 0.1% SDS, 100 mg/ml denatured salmon sperm DNA, and 10% dextran sulfate. Full-length 1.5 kb human E2F1 cDNAs were labeled by the random priming method and used as probes for Northern hybridization. Following hybridization, the membranes were washed twice for five minutes at room temperature in 5 3 SSPE and 0.5% SDS, twice at 378C for 15 minutes in 1 3 SSPE and 0.5% SDS, and once at 378C for 15 minutes in 0.1 3 SSPE and 0.5% SDS and were then exposed to Fuji x-ray film (Fuji Photo Film, Kanagawa, Japan) for 24 to 48 hours at room temperature. The films were analyzed by BASe (Fuji Photo). Cell cycle analysis by fluorescence-activated cell sorter (FACS) Mesangial cells were cultured in 10 cm dishes. The cells were incubated in starved medium for 48 hours before adenovirus infection. After removal of the medium, 109 pfu/ml AdE2F1 solutions were added to the culture dishes for one hour with shaking every 15 minutes, and the cells were then incubated in 0.25% FCS medium for the indicated times. The samples were washed twice with PBS and resuspended in 70% ethanol for at least 12 hours at 48C, and then the fixed and permeabilized cells were collected by centrifugation and were washed with PBS. The cells were denatured by RNase, stained with propidium iodide, and analyzed by flow cytometry using a FACS Calibur (Becton Dickinson, San Jose, CA, USA). The percentages of the cells in G1, S, and G2/M phases were then determined. Measurement of caspase 3 activities A Caspase 3 Fluorometric Protease Assay Kit (MBL, Tokyo, Japan) was used for measurement of caspase 3 activities. In brief, cells were plated in six-well dishes, cultured in starved medium for 48 hours, transfected by adenovirus, and incubated in starved medium that was changed once daily. At indicated times, the cells were collected and lyzed in lysis buffer, and the protein concentration was normalized by the Bradford assay. The lysates were incubated with the same amount of reaction buffer and 50 mm DEVD-AFC substrate for two hours at 378C. Fluorescence was monitored with an excitation wavelength of 400 nm and an emission wavelength of 505 nm.
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Transient transfections and luciferase assays Transfection assays were performed in rat mesangial cells by electroporation methods. Mesangial cells were grown in 20% FCS containing medium until 80% confluence and were then incubated in starved medium for 48 hours to stop the cell cycle. After stimulating the cells with FCS-containing medium for 24 hours, they were washed by PBS, collected by trypsinization, and centrifuged and washed twice by PBS. The cells were put in 0.4 cm cuvettes with 5 mg luciferase reporter plasmid and 5 mg b-galactociderse reporter plasmid, and then they were transfected by electroporation at 300 V using a Bio-Rad gene pulser (Bio-Rad, Hercules, CA, USA). Luciferase reporter plasmids driven by the human cyclin D1 promoter were provided by Dr. M. Eilers. The human cyclin E promoter was provided by Dr. K. Ohtani, and the human cyclin A promoter was provided by Dr. J. Sobczak-Thepot. The cells were incubated in FCS-containing medium for 24 hours and infected by adenovirus. Luciferase activity was measured as described previously 12 hours after infection. CDK4 and CDK2 kinase assay The immune complex kinase assay was performed using essentially the same methods described by Matsushime et al [32]. The mesangial cells were incubated with serumstarved medium for 48 hours. The cells were infected with 109 pfu/ml AdE2F1 or were stimulated by 20% FCS as a positive control. For CDK4 and CDK2 kinase assays, the lysates were prepared 8 and 12 hours after infection, respectively. Immune complexes were recovered by centrifugation after incubating the cell lysates for two hours at 48C with 10 ml of anti-cyclin D1 antibody or anti-cyclin E antibody (Santa Cruz Biotechnology Inc.) and then one hour with 30 ml of protein G-plus agarose (Oncogene Science, Uniondale, NY, USA). Immunoprecipitated proteins were suspended in 30 ml of kinase buffer [50 mm HEPES (pH 7.5), 10 mm MgCl2, 1 mm DTT] containing substrate [0.2 mg of soluble glutathione S-transferasepRb fusion protein (Santa Cruz Biotechnology Inc.) for CDK4 kinase assay, and Histione H1 (Boeringer Mannheim, Germany) for CDK2 assay] and 2.5 mm EGTA, 10 mm b-glycerophosphate, 1 mm NaF, 20 mm ATP, and 10 mCi of [g-32P] ATP (NEN, DuPont, Boston, MA, USA). After incubation for 30 minutes at 308C with occasional mixing, the samples were boiled in polyacrylamide gel sample buffer containing sodium dodecyl sulfate and separated by electrophoresis. Phosphorylated proteins were analyzed by BAS station. Statistical analysis The results were given as means 6 sem. The significance of difference was tested using analysis of variance in multiple comparisons. P , 0.05 was considered statistically significant.
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Fig. 1. (A) Increase of the number of mesangial cells after fetal calf serum (FCS) stimulation. The mesangial cells were incubated in starved medium for 48 hours and were then stimulated by replacement with 20% serum-containing medium. After indicated times, the cells were incubated with WST-8 for two hours. Absorbance that reflected the number of living cells was measured. Each point represents the mean 6 sem of four experiments. *P , 0.05 vs. control; **P , 0.05 vs. 18 hours by analysis of variance (ANOVA) test. (B) Time course of [3H]-thymidine incorporation after serum stimulation. The mesangial cells were treated as described in (A). After indicated times, the cells were incubated with [3H]-thymidine for two hours. Each bar represents the mean 6 sem of four experiments. *P , 0.05 vs. control by ANOVA.
RESULTS Time course of the mesangial cell cycle To characterize mesangial cell proliferation, we examined cell number and DNA synthesis after growth stimulation. Mesangial cells were made quiescent by incubation in starved medium for 48 hours and then stimulated by replacement in medium containing 20% FCS. After the indicated times, the cells were incubated with WST-8 for two hours, and then the absorbance that reflects the surviving cell number was measured. Figure 1A shows that the number of mesangial cells increased by about twofold at 18 hours. This data indicated that the M phase started between 12 and 18 hours after FCS stimulation in our condition. We examined [3H]-thymidine incorporation after FCS stimulation (Fig. 1B). The mesangial cells were treated as described earlier in this article and then incubated with [3H]-thymidine for two hours. [3H]thymidine incorporation was significantly increased 6 hours after FCS stimulation and reached at its peak after 12 hours. These data suggested that almost all of the mesangial cells entered the S phase at some point between 6 and 12 hours and exited the S phase at some point between 12 and 18 hours. To be brief, the majority
of mesangial cells pass through the G1/S phase at some point between 6 and 12 hours and then pass through the G2/M phase at some point between 12 and 18 hours. Expression of the E2F family in rat mesangial cells To determine which member of the E2F family is important and involved in mesangial cell proliferation, the time course of protein expression of the E2F family after FCS stimulation was examined, and it was compared with the mesangial cell cycle discussed earlier in this article. Mesangial cells were rendered quiescent and stimulated by FCS using the same methods described in Figure 1. Nuclear protein was extracted at indicated times. As shown in Figure 2A, protein expression of E2F1 through E2F3 was induced by FCS stimulation. The expression of E2F1 was almost undetectable in a quiescent state and was only slightly detectable at six hours after FCS stimulation. It strongly increased from 12 hours after FCS stimulation. This time course was consistent with the result of [3H]-thymidine incorporation. Although protein expression of E2F2 and E2F3 resembled that of E2F1, magnitudes of their changes were notably smaller than that of E2F1 expression. The increases of E2F2 and E2F3 were obscure compared
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Fig. 3. Time course of E2F1 overexpression after AdE2F1 infection. The mesangial cells were incubated in starved medium for 48 hours and infected with 109 pfu/ml AdE2F1. Nuclear protein was extracted after indicated times. Western blotting was performed with same amount of nuclear protein.
Fig. 2. (A) Induction of E2F family (E2F1 through E2F4) protein expression after serum stimulation in rat mesangial cells. Western blot analysis was performed with a specific antibody for E2F family individuals. Primary cultured rat mesangial cells were incubated in starved medium for 48 hours to render them quiescent. Nuclear protein was extracted at indicated times after stimulation with 20% FCS. Aliquots of the nuclear protein were electrophoresed on a SDS-polyacrylamide gel and visualized by the ECL chemiluminescence system. (B) Induction of E2F1 mRNA expression after stimulation in rat mesangial cells. The mesangial cells were incubated in starved medium for 48 hours to render them quiescent. Total RNA was extracted at indicated times after stimulation with 20% FCS extracted as described in the Methods section. Aliquots of total RNA (20 mg per lane) were electrophoresed on a 1% agarose gel. The membrane was hybridized sequentially using a specific cDNA probe for E2F1. Ribosomal RNA (18S) is shown as a control for RNA loading.
with the increase of E2F1 in the G1/S phase transition. In contrast, protein expression of E2F4 was highest among the E2F family in the quiescent state, but it showed only a slight increase during FCS stimulation. The expression of E2F4 increased at six hours. E2F4 appeared to be mainly expressed in cytosols without growth simulation [33, 34]. When we extracted nuclear protein to detect slight changes after growth stimulation, it was expected that E2F4 expression at zero hours, including cytosolic fraction, would exceed the expression we detected here. Some studies showed E2F4 did not change the amount of expression through the cell cycle and had no influence in promoting the cell cycle [35]. These data suggest that the E2F1, E2F2, and E2F3 correlate with mesangial cell proliferation. The peak of expression of E2F3 at 18 hours was late for a peak in DNA synthesis. In fact, which protein, E2F1 or E2F2, plays a more important role in the mesangial cell cycle could
not be distinguished. We focused on E2F1 in the following study because the E2F1 expression exceeded E2F2 and E2F3 expression during cell cycle progression in mesangial cells. The time course of the expression of E2F1 mRNA was also examined. Figure 2B shows that E2F1 mRNA increased moderately at three hours after FCS stimulation and increased strongly at 12 hours. The time course of mRNA expression was almost the same as that of protein expression. The similarity between these time courses suggested that the transcriptional regulation of E2F1 is important in the mesangial cell cycle. Regulation of cell cycle by E2F1 overexpression in rat mesangial cells Adenovirus-mediated gene transfer was used to investigate whether the overexpression of E2F1 could promote cell cycle progression. In our previous study, we demonstrated the efficiency of transfection and the toxicity by means of adenovirus-mediated transfection to mesangial cells [6]. At first the time course of E2F1 protein expression was examined after AdE2F1 infection. The quiescent mesangial cells were infected with 109 pfu/ml AdE2F1. Then the mesangial cells were harvested at indicated times and the nuclear protein extracted. Figure 3 shows that E2F1 expression significantly increased 6 hours after infection and continued thereafter up to at least 30 hours. We investigated whether only E2F1 overexpression could promote DNA synthesis and cell cycle progression. The quiescent mesangial cells were infected by various concentrations of AdE2F1 for 24 hours, and then the [3H]-thymidine incorporation was measured. As shown in Figure 4, overexpression of E2F1 in rat mesangial cells increased thymidine uptake to 220% in the absence of FCS stimulation, which is an almost equal level to that induced by 20% FCS stimulation. Next, the mesangial cell cycle progression was examined using flow cytometry. Mesangial cells were infected with 109 pfu/ml AdE2F1, after indicated times harvested,
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Fig. 4. Effect of E2F1 overexpression on mesangial cell proliferation as assessed by [3H]-thymidine incorporation. Quiescent mesangial cells cultured in starved medium for 48 hours were incubated with indicated dosages of AdE2F1. Control mesangial cells were infected with 109 pfu/ml AdLacZ. Twenty-four hours after incubation, they were pulsed with [3H]thymidine for four hours. Each bar represents the mean 6 sem of four experiments. *P , 0.05 vs. control by analysis of variance test.
and were investigated to determine the amount of DNA content. Figure 5A illustrates an experimental plotting of the cell number as a function of DNA content at 0, 12, and 24 hours. The data, summarized in Figure 5B, revealed that overexpression of E2F1 alone significantly increased the percentage of the S phase and decreased the percentage of the G0/G1 phase. The percentage of the G2/M phase was increased by E2F1 overexpression, although it is not statistically significant. These data suggest that mesangial cells progress from the G1 phase to the S phase at same point from six hours after E2F1 overexpression and then onward to the M phase at same point from 18 hours. We concluded that overexpression of E2F1 alone could accelerate the G1/S phase transition and promote mesangial cell proliferation. In other words, E2F1 upregulation is sufficient for rat mesangial cell proliferation in certain conditions. Induction of apoptosis by E2F1 overexpression in rat mesangial cells Some studies reported that overexpression of E2F1 induced apoptosis after the induction of the S phase
Fig. 5. Cell cycle analysis using flow cytometry after E2F1 overexpression. Mesangial cells were brought to quiescence as described in Figure 3, were infected with 109 pfu/ml AdE2F1, and subsequently collected at indicated times. The cells were stained with propidium iodine and processed for flow cytometry. (A) Representative tracing. The horizontal axis reflects relative DNA content and the vertical axis represents cell number. (B) The percentage of gated control and AdE2F1-infected cells in each phase of the cell cycle was graphed from the flow cytometric analysis. Each bar represents the mean 6 sem of four experiments. *P , 0.05 vs. 0 hours by ANOVA.
[36–39]. We observed the cell viability over a long time course after the overexpression of E2F1. Mesangial cells were cultured in starved medium to maintain quiescence for two days. Next, we transfected cells with 109 pfu/ml AdE2F1 or with the same amount of AxCALacZ as a control. After the indicated times, we incubated the cells with WST-8 for two hours and then measured the absorbance that reflects the surviving cell number. Figure 6A showed a twofold increase in the number of living mesangial cells 24 hours after E2F1 overexpression. Thereafter, the number of living mesangial cells quickly decreased from 48 hours, whereas the number of control cells gradually increased up to 72 hours. Caspase 3 protease activity was measured to determine whether this decrease from 48 hours reflected apoptosis. The mesangial cells were treated in the same manner described in Figure 6A. After the indicated times, cell lysates were prepared and normalized by protein concentration.
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Figure 6A. These data suggested that E2F1 overexpression completed the mesangial cell cycle and doubled the number of living cells. After that, E2F1 overexpression induced apoptosis.
Fig. 6. (A) Change the number of living mesangial cells after E2F1 overexpression. The mesangial cells were incubated for 48 hours in starved medium. After that, the cells were infected with 109 pfu/ml AdE2F1 (———) or AxCALacZ (- - - - -) as a control. The starved medium was changed every day. After the indicated times, the cells were incubated with WST-8 for two hours. The absorbance that reflects the number of surviving cells was measured. Each point represents the mean 6 sem of three experiments. (B) Induction of caspase 3-like protease activity by E2F1 overexpression. The mesangial cells were treated as described in (A) and were infected with 109 pfu/ml AdE2F1 (j) or AxCALacZ ( ) as a control. After indicated times, cell lysates were prepared and normalized by protein concentration. Caspase 3-like protease activity was measured by proteolytic cleavage of the specific fluorogenic substrate DEVD-AFC. Each bar represents the mean 6 sem of three experiments.
Caspase 3-like protease activity was measured by proteolytic cleavage of the specific fluorogenic substrate DEVD-AFC. Caspase 3-like protease activity was significantly increased at 48 and 72 hours compared with control, but it was lower than control at 24 hours. This increase was consistent with the time course showed in
Induction of the G1 cyclins, cyclin D1, cyclin E, and cyclin A by E2F1 overexpression To elucidate the mechanisms of E2F1-induced mesangial cell cycle progression, we investigated the interaction between E2F1 and G1 cyclins. We previously reported that G1 cyclins play an important role in the mesangial cell cycle [6, 9]. These G1 cyclins are well known to have E2F binding sites in their promoter region [22, 24–26]. However, the network of G1 cyclins and E2F1 has not been clear, and the interaction between G1 cyclins and E2F has not been investigated in primary cultured mesangial cells. To clear the regulation of G1 cyclins and E2F1 in detail, we studied the promoter activity and protein expression of G1 cyclins after E2F1 overexpression. We transfected plasmids containing the cyclin D1, cyclin E, or cyclin A promoter region with luciferase reporter gene to rat mesangial cells by electroporation methods, and then E2F1 was overexpressed using adenovirus vector. Figure 7A showed that E2F1 overexpression significantly increased cyclin D1, cyclin E, and cyclin A promoter activity 5.7-fold, 30.8-fold, and 12.8-fold, respectively. Figure 7B showed the time course of G1 cyclin protein expressions after AdE2F1 infection. The quiescent mesangial cells were infected with AdE2F1 in starved medium. Surprisingly, overexpression of E2F1 caused a sequential induction of three G1 cyclins identical to a sequential induction triggered by growth stimulation. E2F1 increased cyclin D1 from 6 hours up to a peak at 12 hours, and it increased cyclin E and cyclin A from 12 and 18 hours, respectively. We also performed quantitative analysis of protein expressions of G1 cyclins (Fig. 7C). This result was consistent with the time course of flow cytometry analysis. The mesangial cells entered the S phase from 6 hours after E2F1 overexpression and exited the S phase from 18 hours. We examined whether this induction of G1 cyclins contributed to the increased kinase activities of CDK4 and CDK2, respectively. Eight hours of AdE2F1 infection increased CDK4 kinase activity to a level almost equal to that induced by eight hours of FCS stimulation. (Fig. 8, upper panel). CDK2 activity was also increased 2.8 times by E2F1 overexpression for 12 hours (Fig. 8, lower panel), but it was only minimally increased by E2F1 overexpression for eight hours (data not shown). These data suggest that E2F1 initiates G1/S phase transition not only by induction of genes required for DNA synthesis, but also by induction G1 cyclins. E2F1 and G1 cyclins strongly cooperate in promoting the G1/S phase transition in mesangial cells.
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Fig. 7. (A) Effects of E2F1 overexpression on the transcriptional activity of the cyclin D1, cyclin E, and cyclin A promoters. Mesangial cells were equilibrated to the cell cycle phase and subsequently grown in serum containing medium as described in the Methods section. The cells were transfected with cyclin D1, cyclin E, or cyclin A promoter plasmids containing luciferase reporter gene by the electroporation method. After an additional 24 hours of growth, the cells were infected with indicated dosages AdE2F1 or AxCALacZ (control). The cells were collected and assayed for luciferase activities. The results represent the means 6 sem of relative luciferase activities (after collection by protein concentration) from four experiments. (B and C) Effect of E2F1 overexpression on the induction of the cyclin D1 (r), cyclin E (j), and cyclin A (m) proteins. Mesangial cells were brought to quiescence by 48hour serum withdrawal and infected with 109 pfu/ml AdE2F1. Nuclear proteins were extracted indicated times after infection. Aliquots of the nuclear protein were separated on a SDS-PAGE and visualized by the ECL chemiluminescence system (B). Quantitative analysis of protein expressions of G1 cyclins was performed using DNAquant (C).
DISCUSSION In this study, we demonstrated for the first time that E2F expression was a necessary and sufficient condition for mesangial cell proliferation. Moreover, the same case may hold true in many other cell lines as well. The p16/ Rb/E2F regulatory pathway is one of the most frequent targets of genetic alterations in human cancers [40]. This suggests that the regulation of this pathway differs in carcinoma cells and primary culture cells. Several recent studies showed that E2F and G1 cyclins were effective therapeutic targets of mesangial proliferative disease [20, 21, 41]. We therefore thought it was important to clarify the regulation of mesangial cell cycle by E2F for future therapeutic use. We showed that E2F1, E2F2, and E2F3 expressions were increased by serum stimulation, whereas E2F4 expression was not (Fig. 2A). E2F1, E2F2, and E2F3 are well known to be distinctly different from E2F4. Namely, the former stimulate cell cycle progression, whereas the latter does not [42]. However, there are no clear differences between E2F1, E2F2, and E2F3 in terms of their ability to induce cell cycle progression. In mesangial cells,
the peak in E2F3 expression was late for S phase, and E2F1 and E2F2 expressions increased over a time course consistent with that of G1/S phase transition. E2F1 expression increased 20-fold from 6 to 12 hours, whereas E2F2 expression increased by no more than twofold. We presumed that E2F1 was a major protein among the E2F family in mesangial cell cycle progression. E2F1 overexpression increased [3H]-thymidine incorporation and the percentage of cells in the S phase. In our flow cytometry study, E2F1 overexpression tended to increase the percentage of cells in the G2/M and G0/G1 phases at 24 hours, with a twofold increase in the number of living cells at that time point. The percentage of cells that progressed to S and G2/M phases was smaller than the percentages previously reported in other cell lines. We recognize that this discrepancy may be due to the tendency of mesangial cells to stay quiescent, the high expression of cyclin-dependent kinase inhibitor [28], or the efficiency of the gene transfer to the primary culture cell in our system. In any case, these data suggested that E2F1 overexpression could induce the G1/S phase transition and complete mitosis. Mesangial cells have been
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Fig. 8. Induction of CDK4 and CDK2 kinase activity by E2F1 overexpression. The mesangial cells were incubated with starved medium for 48 hours and then either infected with 109 pfu/ml AdE2F1 or stimulated by FCS as a positive control. After 8 hours for the CDK4 kinase assay and 12 hours for the CDK2 kinase assay, cell lysates were immunoprecipitated with anti-cyclin D1 antibody and anti-cyclin E antibody, respectively. Kinase assays were performed with Rb protein or Histone H1 as a substrate for CDK4 and CDK2, respectively.
rarely investigated to determine whether the introduction of an exogenous gene facilitates the cell cycle. To our knowledge, this is the first report in which the expression of a single gene promotes cell cycle re-entry and completes mitosis in mesangial cells. It is well known that E2F1 overexpression causes apoptosis [36–39]. The mechanisms of E2F1-induced apoptosis have been reported to act in both p53-dependent and p53-independent manners. Although it remains unclear precisely how E2F1 induces apoptosis, the process is not simply an artifact of protein overexpression. The induction of apoptosis is specific to E2F1 among the E2F family, and neither E2F2 nor E2F3 have been showed to have any connection with apoptosis [43]. In mesangial cells, E2F1 overexpression induced apoptosis at 48 hours after infection. At this time point, mitosis was completed once. Caspase 3 activity was also increased at 48 hours. We speculate that E2F1 overexpression induces proper cell division, whereas continuous E2F1 expression or E2F1-induced gene expression at an inappropriate phase causes apoptosis. Some studies reported that apoptosis was increased in the progression of glomerular sclerosis. Further studies are needed to elucidate the relationship between progression of glomerular sclerosis and E2F1induced apoptosis. Various experiments suggest that free E2F increases the expression of genes required for DNA synthesis, as well as regulatory genes such as c-myc, N-myc, erb-B, c-myb, and B-myb [44–50]. It is also well known that cyclin D1, cyclin E, and cyclin A have E2F binding sites in their promoter regions [22, 24–26]. Although it is established that free E2F induces the expression of cyclin E and cyclin A [24, 26], E2F has not been reported to
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increase the protein level of cyclin D1. Watanabe et al reported that E2F1 overexpression decreased cyclin D1 expression dependent on the Sp1 binding site [51]. Furthermore, Ohtani, Degregori, and Nevins showed that E2F1 overexpression activated cyclin D1 promoter without increasing the protein level [24]. Fan and Bertino suggested that there may be a positive feedback regulation loop between E2F and cyclin D1 by means of a dominant negative mutant of E2F1 [52]. In our data, E2F1 overexpression increased both the promoter activity and protein expression of cyclin D1. Our study is the first to report an interaction between E2F and G1 cyclins in primary cultured cells, and we postulate that the difference in cyclin D1 regulation induced by E2F1 reflects a difference in the mechanisms that regulate G1/S phase transition between primary cultured cells and immortal cells. In our preliminary experiments, E2F1 overexpression increased cyclin D1 promoter activity but not protein expression in LLC-PK1 cells (data not shown). Cyclin D1/CDK4 and cyclin E/CDK2 complexes facilitate phosphorylation of the pRb family with the release of free E2F, and then the released E2F increases G1 cyclins and promotes G1/S phase progression. E2F and G1 cyclins form the firm, positive feedback loop. Late induction of cyclin A may contribute to the reduction of DNA-binding activities of the E2F1-DP-1 dimer [53–55], in other words, form negative feedback loop. Various studies have demonstrated that the expressions of specific cyclins and CDKs and CDK inhibitors change during mesangial cell proliferation in cell culture systems [5–9] and in in vivo experimental models [10]. Some studies indicated that inhibition of CDKs or E2F suppressed mesangial cell proliferation [20, 21, 41]. Smooth muscle cell proliferation after endothelium injury was suppressed by infusion of an E2F decoy oligonucleotide [56]. These experiments suggest new therapeutic approaches to benign proliferative disease. Because most of our knowledge about cell cycle regulation is based on experiments using cells derived from carcinoma, it is important to clarify the difference in the cell cycle regulation between primary culture cells and cell lines. Studies of cell cycle regulatory proteins, including cyclins and E2F in human renal disease, have not yet been reported. Mesangial cell proliferation continues for a few decades in human chronic glomerulonephritis, and it is not clear whether cell cycle regulatory genes such as cyclins and E2F are involved in the pathogenesis of this disease. Further studies are needed to determine whether these cell cycle control mechanisms are involved in such a long-term proliferation. ACKNOWLEDGMENTS We thank for Dr. I. Saito for kindly providing AxCALacZ, and Drs. J.R. Nevins and K. Ohtani for kindly providing AdE2F1 and cyclin E promoter. We are also grateful to Drs. M. Eilers and J.
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Sobczak-Thepot for kindly providing cyclin D1 and cyclin A promoter, respectively. Reprint requests to Yoshio Terada, M.D., Second Department of Internal Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: [email protected]
APPENDIX Abbreviations used in this article are: ATP, adenosine 59-triphosphate; AdE2F1, replication-defective recombinant adenovirus expressing E2F1; AxCALacZ, recombinant adenovirus expressing the LacZ gene; BSA, bovine serum albumin; CDK, cyclin-dependent protein kinase; CMV, cytomegalovirus; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetatic acid; EGTA, egtazic acid; FCS, fetal calf serum; PBS, phosphate buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; WST-8, tetrazolium monosodium salt.
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