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Comparative Study
. 2000 May;20(10):3417-24.
doi: 10.1128/MCB.20.10.3417-3424.2000.

E2F4 and E2F1 have similar proliferative properties but different apoptotic and oncogenic properties in vivo

D Wang  1 , J L Russell, D G Johnson
Affiliations
Comparative Study

E2F4 and E2F1 have similar proliferative properties but different apoptotic and oncogenic properties in vivo

D Wang et al. Mol Cell Biol. 2000 May.

Abstract

Loss of retinoblastoma (Rb) tumor suppressor function, as occurs in many cancers, leads to uncontrolled proliferation, an increased propensity to undergo apoptosis, and tumorigenesis. Rb negatively regulates multiple E2F transcription factors, but the role of the different E2F family members in manifesting the cellular response to Rb inactivation is unclear. To study the effect of deregulated E2F4 activity on cell growth control and tumorigenesis, transgenic mouse lines expressing the E2F4 gene under the control of a keratin 5 (K5) promoter were developed, and their phenotypes were compared to those of previously generated K5 E2F1 transgenic mice. In contrast to what has been observed in vitro, ectopically expressed E2F4 was found to localize to the nucleus and induce proliferation to an extent similar to that induced by E2F1 in transgenic tissue. Unlike E2F1, E2F4 does not induce apoptosis, and this correlates with the differential abilities of these two E2F species to stimulate p19(ARF) expression in vivo. To examine the role of E2F4 in tumor development, the mouse skin two-stage carcinogenesis model was utilized. Unlike E2F1 transgenic mice, E2F4 transgenic mice developed skin tumors with a decreased latency and increased incidence compared to those characteristics in wild-type controls. These findings demonstrate that while the effects of E2F1 and E2F4 on cell proliferation in vivo are similar, their apoptotic and oncogenic properties are quite different.

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Figures

FIG. 1
FIG. 1
Generation of K5 E2F4 transgenic lines. (A) Schematic representation of the K5 E2F4 transgene. Primary keratinocytes were isolated and cultured from the epidermis of newborn nontransgenic (wild-type) and K5 E2F4 transgenic mice (lines 4.0 and 4.3). (B) Total RNA (20 μg per lane) from primary keratinocytes was subjected to Northern blot analysis using the human E2F4 cDNA as a probe. (C) Whole-cell protein lysate (20 μg per lane) from primary-keratinocyte cultures was subjected to Western blot analysis using polyclonal antiserum specific for E2F4.
FIG. 2
FIG. 2
Ectopically expressed E2F4 localizes to the nucleus in transgenic tissue. Tail sections were taken from nontransgenic (A) or K5 E2F4 line 4.0 (B) mice and immunohistochemically stained with antiserum specific for E2F4. BL, basal layer; HF, hair follicle. (C) Higher magnification of K5 E2F4 line 4.0 tissue demonstrating the nuclear localization of E2F4.
FIG. 3
FIG. 3
E2F DNA-binding activity from K5 E2F4 transgenic keratinocytes. (A) An E2F EMSA was performed using whole-cell extracts (10 μg) from primary keratinocytes isolated from nontransgenic (lanes 1 and 2), line 4.0 (lanes 3 and 4), or line 4.3 mice (lanes 5 and 6). Antiserum specific for E2F4 was added (lanes 2, 4, and 6) to identify complexes containing E2F4. (B) An E2F EMSA was performed using whole-cell extract (10 μg) from primary keratinocytes isolated from line 4.0 mice. Excess double-stranded oligonucleotide (20 ng) containing either wild-type (wt) E2F sites (lane 1) or mutated (mut) E2F sites (lane 2) was added to the binding reaction mixtures to distinguish specific E2F complexes. Antisera specific for E2F4 (lane 3), Rb (lane 4), p107 (lane 5), and p130 (lane 6) were added to identify proteins in specific complexes.
FIG. 4
FIG. 4
Histological appearance of K5 E2F4 transgenic skin. Photomicrographs of skin samples from a nontransgenic mouse (A) and K5 E2F4 transgenic line 4.0 (B) and line 4.3 (C) mice stained with hematoxylin and eosin.
FIG. 5
FIG. 5
Hyperplasia, proliferation, and apoptosis in K5 E2F1 and K5 E2F4 transgenic epidermis. (A) Epidermal thickness was measured from skin samples taken from nontransgenic (wild-type [wt]), K5 E2F4 line 4.0 (4.0), K5 E2F4 line 4.3 (4.3), K5 E2F1 line 1.1 (1.1), and K5 E2F1 line 1.0 (1.0) mice. Samples were taken from five different mice in each group, and 100 measurements were taken for each sample to calculate the average thickness. (B) The percentage of interfollicular basal keratinocytes in S phase was calculated for the same mice as those used to obtain the results in panel A by measuring BrdU incorporation. Mice were injected with BrdU 30 min prior to sacrifice, and antibody specific for BrdU was used to immunostain skin samples. Five hundred cells were counted per sample, and the average percentages of positive cells from five mice in each group are presented. (C) The TUNEL assay was used to examine apoptosis in skin sections from the same mice. At least 40 measurements were taken from each sample (five mice per group) to calculate the average number of TUNEL-positive epidermal cells per 10 mm of linear skin.
FIG. 6
FIG. 6
Induction of p19ARF and cyclin E expression in K5 E2F1 but not K5 E2F4 keratinocytes. Total RNA (20 μg per lane) was isolated from primary keratinocytes derived from nontransgenic (wild-type [wt]), K5 E2F4 line 4.0 (4.0), K5 E2F4 line 4.3 (4.3), and K5 E2F1 line 1.0 (1.0) transgenic mice. Northern blot analysis was performed on the same filter using probes for murine p19ARF, cyclin E, and 7S.
FIG. 7
FIG. 7
Response of K5 E2F4 transgenic mice to TPA. K5 E2F4 line 4.3 transgenic mice (E2F4.3) and nontransgenic siblings (wild type) were treated with 2 μg of TPA or acetone vehicle control (ACE) twice weekly for 2 weeks. Mice were sacrificed 24 h following the last TPA treatment and 30 min following BrdU injection. (A) The average percentage of basal keratinocytes in S phase was determined for anti-BrdU immunostained skin sections from three mice in each group. (B) The average epidermal thickness was determined from hematoxylin- and eosin-stained skin sections from three mice in each group.
FIG. 8
FIG. 8
Enhanced tumor development in K5 E2F4 transgenic mice following two-stage carcinogenesis. (A) Carcinogenesis was initiated in mice from both K5 E2F4 transgenic lines (E2F4.0+ and E2F4.3+) and nontransgenic siblings (E2F4.0− and E2F4.3−) by topical application of 10 nmol of DMBA to shaved dorsal skin. Two weeks following initiation, tumors were promoted by twice weekly applications of TPA (1 μg) to dorsal skin for 20 weeks. The average numbers of palpable tumors per mouse at each week are presented. The number of mice in each group was five for line 4.0+, 8 for line 4.0−, six for line 4.3+, and three for line 4.3−. (B) The size of each tumor in the study was calculated at 18 weeks of promotion by multiplying tumor length by tumor width. The average sizes of tumors from nontransgenic (39 tumors total), line 4.0 (61 tumors total), and line 4.3 (89 tumors total) mice are presented.
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