p27(Kip1) induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus and hepatoma cells

Abstract

The Ah receptor (AhR), a bHLH/PAS transcription factor, mediates dioxin toxicity in the immune system, skin, testis and liver. Toxic phenomena are associated with altered cell proliferation or differentiation, but signaling pathways of AhR in cell cycle regulation are poorly understood. Here we show that AhR induces the p27(Kip1) cyclin/cdk inhibitor by altering Kip1 transcription in a direct mode without the need for ongoing protein synthesis or cell proliferation. This is the first example of Kip1 being a direct transcriptional target of a toxic agent that affects cell proliferation. Kip1 causes dioxin-induced suppression of 5L hepatoma cell proliferation because Kip1 antisense-expressing cells are resistant to dioxins. Kip1 is also induced by dioxins in cultures of fetal thymus glands concomitant with inhibition of proliferation and severe reduction of thymocyte recovery. Kip1 expression is likely to mediate these effects as thymic glands of Kip1-deficient mice (Kip1(Delta51)) are largely, though not completely, resistant.

Figure 1

Transcription-dependent delay of cell cycle progression by TCDD. (A) Asynchronously growing 5L cells were starved by serum deprivation or treated with 1 nm TCDD for 48 hr. The degree of Rb phosphorylation was detected by Western blot analysis in which the antibody apparently preferentially recognizes P-Rb. (B) 5L Cells were growth arrested by starvation in serum-free medium for 24 hr and synchronously induced to proliferate by addition of serum. Cell cycle progression was monitored 8 hr later by the phosphorylation status of Rb as in A. Cells were treated 2 hr prior to analysis with 1 nm TCDD, the transcriptional inhibitor actinomycin D (5 μg/ml), or both. (C) Wild-type and mutant AhR (mAhR) were expressed together with GFP in AhR-deficient BP8 cells to reconstitute TCDD-induced cell cycle arrest. A schematic outline of AhR mutations is shown. Proliferation of TCDD-treated cells was determined by flow cytometry after DNA staining with H33258 as the percentage of cells in the G₂ and S phases of the cell cycle. Efficiently transfected green fluorescing cells ( ± AhR/mAhR) were compared with the cells that were not efficiently transfected (−AhR) from the same culture dish and FACS analysis. Similar results were obtained in two to six independent experiments.

Figure 1

Transcription-dependent delay of cell cycle progression by TCDD. (A) Asynchronously growing 5L cells were starved by serum deprivation or treated with 1 nm TCDD for 48 hr. The degree of Rb phosphorylation was detected by Western blot analysis in which the antibody apparently preferentially recognizes P-Rb. (B) 5L Cells were growth arrested by starvation in serum-free medium for 24 hr and synchronously induced to proliferate by addition of serum. Cell cycle progression was monitored 8 hr later by the phosphorylation status of Rb as in A. Cells were treated 2 hr prior to analysis with 1 nm TCDD, the transcriptional inhibitor actinomycin D (5 μg/ml), or both. (C) Wild-type and mutant AhR (mAhR) were expressed together with GFP in AhR-deficient BP8 cells to reconstitute TCDD-induced cell cycle arrest. A schematic outline of AhR mutations is shown. Proliferation of TCDD-treated cells was determined by flow cytometry after DNA staining with H33258 as the percentage of cells in the G₂ and S phases of the cell cycle. Efficiently transfected green fluorescing cells ( ± AhR/mAhR) were compared with the cells that were not efficiently transfected (−AhR) from the same culture dish and FACS analysis. Similar results were obtained in two to six independent experiments.

Figure 1

Transcription-dependent delay of cell cycle progression by TCDD. (A) Asynchronously growing 5L cells were starved by serum deprivation or treated with 1 nm TCDD for 48 hr. The degree of Rb phosphorylation was detected by Western blot analysis in which the antibody apparently preferentially recognizes P-Rb. (B) 5L Cells were growth arrested by starvation in serum-free medium for 24 hr and synchronously induced to proliferate by addition of serum. Cell cycle progression was monitored 8 hr later by the phosphorylation status of Rb as in A. Cells were treated 2 hr prior to analysis with 1 nm TCDD, the transcriptional inhibitor actinomycin D (5 μg/ml), or both. (C) Wild-type and mutant AhR (mAhR) were expressed together with GFP in AhR-deficient BP8 cells to reconstitute TCDD-induced cell cycle arrest. A schematic outline of AhR mutations is shown. Proliferation of TCDD-treated cells was determined by flow cytometry after DNA staining with H33258 as the percentage of cells in the G₂ and S phases of the cell cycle. Efficiently transfected green fluorescing cells ( ± AhR/mAhR) were compared with the cells that were not efficiently transfected (−AhR) from the same culture dish and FACS analysis. Similar results were obtained in two to six independent experiments.

Figure 2

Induction of the p27^Kip1 cell cycle inhibitor during TCDD-dependent delay of cell cycle progression. Biochemical properties of G₁-phase cyclins and cdks were analyzed from 30%–60% confluent asynchronous 5L cell cultures that, except for those in C and G, had been treated for 24 hr with TCDD or DMSO. Cyclin E-dependent histone H1 kinase activity (A) was measured in an immune complex kinase assay using an anti-cyclin E (lanes 1,2) or a nonspecific antibody (lanes 3,4). Amounts of (B) G₁-phase cyclins and cdks, and (C) p27^Kip1 were determined by Western blot analysis. (D) Amounts of cdk2 and Kip1 associated with cyclin E were determined by Western blot analysis in immune precipitates similar to those used in A. (E) The amount of free cyclin E not bound in Kip1-containing protein complexes was determined by Western blot analysis of extracts that had been immunodepleted against Kip1 or a matched nonspecific (NS) antigen (N-CAM). (F) Protein levels of p27^Kip1 were determined by Western blot analysis of whole-cell extracts from solvent- or TCDD-treated cultures of the AhR-expressing 5L wild-type cells or their AhR-deficient BP8^AhR− derivatives. (G) Inducibility of Kip1 protein levels was analyzed in BP8 cells that ectopically expressed AhR (BP8^Ahr+). These BP8^Ahr+ cells (lanes 3,4) had been generated by transient transfection of expression vectors for AhR and a truncated murine MHC protein. TCDD treatment was from 24 hr to 48 hr after transfection. Subsequently, efficiently transfected cells were isolated for Western blot analysis by magnetic cell sorting directed against the expressed MHC protein. Control cultures (lanes 1,2) received only the MHC selection marker and the empty expression vector.

Figure 2

Induction of the p27^Kip1 cell cycle inhibitor during TCDD-dependent delay of cell cycle progression. Biochemical properties of G₁-phase cyclins and cdks were analyzed from 30%–60% confluent asynchronous 5L cell cultures that, except for those in C and G, had been treated for 24 hr with TCDD or DMSO. Cyclin E-dependent histone H1 kinase activity (A) was measured in an immune complex kinase assay using an anti-cyclin E (lanes 1,2) or a nonspecific antibody (lanes 3,4). Amounts of (B) G₁-phase cyclins and cdks, and (C) p27^Kip1 were determined by Western blot analysis. (D) Amounts of cdk2 and Kip1 associated with cyclin E were determined by Western blot analysis in immune precipitates similar to those used in A. (E) The amount of free cyclin E not bound in Kip1-containing protein complexes was determined by Western blot analysis of extracts that had been immunodepleted against Kip1 or a matched nonspecific (NS) antigen (N-CAM). (F) Protein levels of p27^Kip1 were determined by Western blot analysis of whole-cell extracts from solvent- or TCDD-treated cultures of the AhR-expressing 5L wild-type cells or their AhR-deficient BP8^AhR− derivatives. (G) Inducibility of Kip1 protein levels was analyzed in BP8 cells that ectopically expressed AhR (BP8^Ahr+). These BP8^Ahr+ cells (lanes 3,4) had been generated by transient transfection of expression vectors for AhR and a truncated murine MHC protein. TCDD treatment was from 24 hr to 48 hr after transfection. Subsequently, efficiently transfected cells were isolated for Western blot analysis by magnetic cell sorting directed against the expressed MHC protein. Control cultures (lanes 1,2) received only the MHC selection marker and the empty expression vector.

Figure 2

Induction of the p27^Kip1 cell cycle inhibitor during TCDD-dependent delay of cell cycle progression. Biochemical properties of G₁-phase cyclins and cdks were analyzed from 30%–60% confluent asynchronous 5L cell cultures that, except for those in C and G, had been treated for 24 hr with TCDD or DMSO. Cyclin E-dependent histone H1 kinase activity (A) was measured in an immune complex kinase assay using an anti-cyclin E (lanes 1,2) or a nonspecific antibody (lanes 3,4). Amounts of (B) G₁-phase cyclins and cdks, and (C) p27^Kip1 were determined by Western blot analysis. (D) Amounts of cdk2 and Kip1 associated with cyclin E were determined by Western blot analysis in immune precipitates similar to those used in A. (E) The amount of free cyclin E not bound in Kip1-containing protein complexes was determined by Western blot analysis of extracts that had been immunodepleted against Kip1 or a matched nonspecific (NS) antigen (N-CAM). (F) Protein levels of p27^Kip1 were determined by Western blot analysis of whole-cell extracts from solvent- or TCDD-treated cultures of the AhR-expressing 5L wild-type cells or their AhR-deficient BP8^AhR− derivatives. (G) Inducibility of Kip1 protein levels was analyzed in BP8 cells that ectopically expressed AhR (BP8^Ahr+). These BP8^Ahr+ cells (lanes 3,4) had been generated by transient transfection of expression vectors for AhR and a truncated murine MHC protein. TCDD treatment was from 24 hr to 48 hr after transfection. Subsequently, efficiently transfected cells were isolated for Western blot analysis by magnetic cell sorting directed against the expressed MHC protein. Control cultures (lanes 1,2) received only the MHC selection marker and the empty expression vector.

Figure 2

Induction of the p27^Kip1 cell cycle inhibitor during TCDD-dependent delay of cell cycle progression. Biochemical properties of G₁-phase cyclins and cdks were analyzed from 30%–60% confluent asynchronous 5L cell cultures that, except for those in C and G, had been treated for 24 hr with TCDD or DMSO. Cyclin E-dependent histone H1 kinase activity (A) was measured in an immune complex kinase assay using an anti-cyclin E (lanes 1,2) or a nonspecific antibody (lanes 3,4). Amounts of (B) G₁-phase cyclins and cdks, and (C) p27^Kip1 were determined by Western blot analysis. (D) Amounts of cdk2 and Kip1 associated with cyclin E were determined by Western blot analysis in immune precipitates similar to those used in A. (E) The amount of free cyclin E not bound in Kip1-containing protein complexes was determined by Western blot analysis of extracts that had been immunodepleted against Kip1 or a matched nonspecific (NS) antigen (N-CAM). (F) Protein levels of p27^Kip1 were determined by Western blot analysis of whole-cell extracts from solvent- or TCDD-treated cultures of the AhR-expressing 5L wild-type cells or their AhR-deficient BP8^AhR− derivatives. (G) Inducibility of Kip1 protein levels was analyzed in BP8 cells that ectopically expressed AhR (BP8^Ahr+). These BP8^Ahr+ cells (lanes 3,4) had been generated by transient transfection of expression vectors for AhR and a truncated murine MHC protein. TCDD treatment was from 24 hr to 48 hr after transfection. Subsequently, efficiently transfected cells were isolated for Western blot analysis by magnetic cell sorting directed against the expressed MHC protein. Control cultures (lanes 1,2) received only the MHC selection marker and the empty expression vector.

Figure 2

Induction of the p27^Kip1 cell cycle inhibitor during TCDD-dependent delay of cell cycle progression. Biochemical properties of G₁-phase cyclins and cdks were analyzed from 30%–60% confluent asynchronous 5L cell cultures that, except for those in C and G, had been treated for 24 hr with TCDD or DMSO. Cyclin E-dependent histone H1 kinase activity (A) was measured in an immune complex kinase assay using an anti-cyclin E (lanes 1,2) or a nonspecific antibody (lanes 3,4). Amounts of (B) G₁-phase cyclins and cdks, and (C) p27^Kip1 were determined by Western blot analysis. (D) Amounts of cdk2 and Kip1 associated with cyclin E were determined by Western blot analysis in immune precipitates similar to those used in A. (E) The amount of free cyclin E not bound in Kip1-containing protein complexes was determined by Western blot analysis of extracts that had been immunodepleted against Kip1 or a matched nonspecific (NS) antigen (N-CAM). (F) Protein levels of p27^Kip1 were determined by Western blot analysis of whole-cell extracts from solvent- or TCDD-treated cultures of the AhR-expressing 5L wild-type cells or their AhR-deficient BP8^AhR− derivatives. (G) Inducibility of Kip1 protein levels was analyzed in BP8 cells that ectopically expressed AhR (BP8^Ahr+). These BP8^Ahr+ cells (lanes 3,4) had been generated by transient transfection of expression vectors for AhR and a truncated murine MHC protein. TCDD treatment was from 24 hr to 48 hr after transfection. Subsequently, efficiently transfected cells were isolated for Western blot analysis by magnetic cell sorting directed against the expressed MHC protein. Control cultures (lanes 1,2) received only the MHC selection marker and the empty expression vector.

Figure 2

Induction of the p27^Kip1 cell cycle inhibitor during TCDD-dependent delay of cell cycle progression. Biochemical properties of G₁-phase cyclins and cdks were analyzed from 30%–60% confluent asynchronous 5L cell cultures that, except for those in C and G, had been treated for 24 hr with TCDD or DMSO. Cyclin E-dependent histone H1 kinase activity (A) was measured in an immune complex kinase assay using an anti-cyclin E (lanes 1,2) or a nonspecific antibody (lanes 3,4). Amounts of (B) G₁-phase cyclins and cdks, and (C) p27^Kip1 were determined by Western blot analysis. (D) Amounts of cdk2 and Kip1 associated with cyclin E were determined by Western blot analysis in immune precipitates similar to those used in A. (E) The amount of free cyclin E not bound in Kip1-containing protein complexes was determined by Western blot analysis of extracts that had been immunodepleted against Kip1 or a matched nonspecific (NS) antigen (N-CAM). (F) Protein levels of p27^Kip1 were determined by Western blot analysis of whole-cell extracts from solvent- or TCDD-treated cultures of the AhR-expressing 5L wild-type cells or their AhR-deficient BP8^AhR− derivatives. (G) Inducibility of Kip1 protein levels was analyzed in BP8 cells that ectopically expressed AhR (BP8^Ahr+). These BP8^Ahr+ cells (lanes 3,4) had been generated by transient transfection of expression vectors for AhR and a truncated murine MHC protein. TCDD treatment was from 24 hr to 48 hr after transfection. Subsequently, efficiently transfected cells were isolated for Western blot analysis by magnetic cell sorting directed against the expressed MHC protein. Control cultures (lanes 1,2) received only the MHC selection marker and the empty expression vector.

Figure 2

Induction of the p27^Kip1 cell cycle inhibitor during TCDD-dependent delay of cell cycle progression. Biochemical properties of G₁-phase cyclins and cdks were analyzed from 30%–60% confluent asynchronous 5L cell cultures that, except for those in C and G, had been treated for 24 hr with TCDD or DMSO. Cyclin E-dependent histone H1 kinase activity (A) was measured in an immune complex kinase assay using an anti-cyclin E (lanes 1,2) or a nonspecific antibody (lanes 3,4). Amounts of (B) G₁-phase cyclins and cdks, and (C) p27^Kip1 were determined by Western blot analysis. (D) Amounts of cdk2 and Kip1 associated with cyclin E were determined by Western blot analysis in immune precipitates similar to those used in A. (E) The amount of free cyclin E not bound in Kip1-containing protein complexes was determined by Western blot analysis of extracts that had been immunodepleted against Kip1 or a matched nonspecific (NS) antigen (N-CAM). (F) Protein levels of p27^Kip1 were determined by Western blot analysis of whole-cell extracts from solvent- or TCDD-treated cultures of the AhR-expressing 5L wild-type cells or their AhR-deficient BP8^AhR− derivatives. (G) Inducibility of Kip1 protein levels was analyzed in BP8 cells that ectopically expressed AhR (BP8^Ahr+). These BP8^Ahr+ cells (lanes 3,4) had been generated by transient transfection of expression vectors for AhR and a truncated murine MHC protein. TCDD treatment was from 24 hr to 48 hr after transfection. Subsequently, efficiently transfected cells were isolated for Western blot analysis by magnetic cell sorting directed against the expressed MHC protein. Control cultures (lanes 1,2) received only the MHC selection marker and the empty expression vector.

Figure 3

Induction of Kip1 mRNA expression in 5L cells by TCDD. (A) Kip1 mRNA levels were determined by Northern blot analysis in 5L cells after 4 hr exposure to TCDD with or without 30 min of pretreatment with the translational inhibitor cycloheximide (50 μg/ml). Cycloheximide treatment was efficient because p27^Kip1 protein accumulation was prevented (third panel) whereas levels of a noninducible protein (p38^HOG1 kinase) remained unchanged in the same extracts. (B,C) Kip1 mRNA stability in control or TCDD-treated 5L cells was analyzed after induction for 4 hr and disruption of further transcription by actinomycin D (5 μg/ml). The decay of Kip1 mRNA was followed by Northern blot analysis and PhosphorImager quantification in comparison to GAPDH mRNA. mRNA half-life times were determined by regression analysis from the presentation in C. (□) −TCDD; (█) +TCDD. (D) The transcriptional rate of the Kip1 gene was analyzed by a nuclear run-off analysis (Greenberg and Ziff 1984) with nuclei prepared from 5L cells that had been treated for 4 hr or 24 hr with 1 nm TCDD or the DMSO solvent. A representative result (24 hr) is shown. (E) Kip1 promoter reporter gene constructs (Kwon et al. 1996) comprising either 1609, 1382, or 42 bp upstream of the transcriptional start site were stably integrated into 5L cells. Also a Kip1 promoter fragment comprising base pairs −1609 to −1312 in front of a TK₈₁-promoter–luciferase reporter gene (Nordeen 1988) was stably transfected. Inducibility in cell pools (average values of triplicate determinations ± s.d.) was calculated as the ratio of reporter gene activity from cells that had been treated for 24 hr with 1 nm TCDD or the solvent, respectively. (F) Kip1 mRNA expression and inducibility by TCDD were tested in 5L cells that had been serum starved for 24 hr. Cells were treated for additional 4 hr in serum-free medium with 1 nm TCDD or the DMSO solvent prior to Northern blot analysis. Fractions of cells in the S–G₂/M segment of the cell cycle were determined from parallel cultures by flow cytometry. (N.D.) Not determined.

Figure 3

Induction of Kip1 mRNA expression in 5L cells by TCDD. (A) Kip1 mRNA levels were determined by Northern blot analysis in 5L cells after 4 hr exposure to TCDD with or without 30 min of pretreatment with the translational inhibitor cycloheximide (50 μg/ml). Cycloheximide treatment was efficient because p27^Kip1 protein accumulation was prevented (third panel) whereas levels of a noninducible protein (p38^HOG1 kinase) remained unchanged in the same extracts. (B,C) Kip1 mRNA stability in control or TCDD-treated 5L cells was analyzed after induction for 4 hr and disruption of further transcription by actinomycin D (5 μg/ml). The decay of Kip1 mRNA was followed by Northern blot analysis and PhosphorImager quantification in comparison to GAPDH mRNA. mRNA half-life times were determined by regression analysis from the presentation in C. (□) −TCDD; (█) +TCDD. (D) The transcriptional rate of the Kip1 gene was analyzed by a nuclear run-off analysis (Greenberg and Ziff 1984) with nuclei prepared from 5L cells that had been treated for 4 hr or 24 hr with 1 nm TCDD or the DMSO solvent. A representative result (24 hr) is shown. (E) Kip1 promoter reporter gene constructs (Kwon et al. 1996) comprising either 1609, 1382, or 42 bp upstream of the transcriptional start site were stably integrated into 5L cells. Also a Kip1 promoter fragment comprising base pairs −1609 to −1312 in front of a TK₈₁-promoter–luciferase reporter gene (Nordeen 1988) was stably transfected. Inducibility in cell pools (average values of triplicate determinations ± s.d.) was calculated as the ratio of reporter gene activity from cells that had been treated for 24 hr with 1 nm TCDD or the solvent, respectively. (F) Kip1 mRNA expression and inducibility by TCDD were tested in 5L cells that had been serum starved for 24 hr. Cells were treated for additional 4 hr in serum-free medium with 1 nm TCDD or the DMSO solvent prior to Northern blot analysis. Fractions of cells in the S–G₂/M segment of the cell cycle were determined from parallel cultures by flow cytometry. (N.D.) Not determined.

Figure 3

Induction of Kip1 mRNA expression in 5L cells by TCDD. (A) Kip1 mRNA levels were determined by Northern blot analysis in 5L cells after 4 hr exposure to TCDD with or without 30 min of pretreatment with the translational inhibitor cycloheximide (50 μg/ml). Cycloheximide treatment was efficient because p27^Kip1 protein accumulation was prevented (third panel) whereas levels of a noninducible protein (p38^HOG1 kinase) remained unchanged in the same extracts. (B,C) Kip1 mRNA stability in control or TCDD-treated 5L cells was analyzed after induction for 4 hr and disruption of further transcription by actinomycin D (5 μg/ml). The decay of Kip1 mRNA was followed by Northern blot analysis and PhosphorImager quantification in comparison to GAPDH mRNA. mRNA half-life times were determined by regression analysis from the presentation in C. (□) −TCDD; (█) +TCDD. (D) The transcriptional rate of the Kip1 gene was analyzed by a nuclear run-off analysis (Greenberg and Ziff 1984) with nuclei prepared from 5L cells that had been treated for 4 hr or 24 hr with 1 nm TCDD or the DMSO solvent. A representative result (24 hr) is shown. (E) Kip1 promoter reporter gene constructs (Kwon et al. 1996) comprising either 1609, 1382, or 42 bp upstream of the transcriptional start site were stably integrated into 5L cells. Also a Kip1 promoter fragment comprising base pairs −1609 to −1312 in front of a TK₈₁-promoter–luciferase reporter gene (Nordeen 1988) was stably transfected. Inducibility in cell pools (average values of triplicate determinations ± s.d.) was calculated as the ratio of reporter gene activity from cells that had been treated for 24 hr with 1 nm TCDD or the solvent, respectively. (F) Kip1 mRNA expression and inducibility by TCDD were tested in 5L cells that had been serum starved for 24 hr. Cells were treated for additional 4 hr in serum-free medium with 1 nm TCDD or the DMSO solvent prior to Northern blot analysis. Fractions of cells in the S–G₂/M segment of the cell cycle were determined from parallel cultures by flow cytometry. (N.D.) Not determined.

Figure 3

Induction of Kip1 mRNA expression in 5L cells by TCDD. (A) Kip1 mRNA levels were determined by Northern blot analysis in 5L cells after 4 hr exposure to TCDD with or without 30 min of pretreatment with the translational inhibitor cycloheximide (50 μg/ml). Cycloheximide treatment was efficient because p27^Kip1 protein accumulation was prevented (third panel) whereas levels of a noninducible protein (p38^HOG1 kinase) remained unchanged in the same extracts. (B,C) Kip1 mRNA stability in control or TCDD-treated 5L cells was analyzed after induction for 4 hr and disruption of further transcription by actinomycin D (5 μg/ml). The decay of Kip1 mRNA was followed by Northern blot analysis and PhosphorImager quantification in comparison to GAPDH mRNA. mRNA half-life times were determined by regression analysis from the presentation in C. (□) −TCDD; (█) +TCDD. (D) The transcriptional rate of the Kip1 gene was analyzed by a nuclear run-off analysis (Greenberg and Ziff 1984) with nuclei prepared from 5L cells that had been treated for 4 hr or 24 hr with 1 nm TCDD or the DMSO solvent. A representative result (24 hr) is shown. (E) Kip1 promoter reporter gene constructs (Kwon et al. 1996) comprising either 1609, 1382, or 42 bp upstream of the transcriptional start site were stably integrated into 5L cells. Also a Kip1 promoter fragment comprising base pairs −1609 to −1312 in front of a TK₈₁-promoter–luciferase reporter gene (Nordeen 1988) was stably transfected. Inducibility in cell pools (average values of triplicate determinations ± s.d.) was calculated as the ratio of reporter gene activity from cells that had been treated for 24 hr with 1 nm TCDD or the solvent, respectively. (F) Kip1 mRNA expression and inducibility by TCDD were tested in 5L cells that had been serum starved for 24 hr. Cells were treated for additional 4 hr in serum-free medium with 1 nm TCDD or the DMSO solvent prior to Northern blot analysis. Fractions of cells in the S–G₂/M segment of the cell cycle were determined from parallel cultures by flow cytometry. (N.D.) Not determined.

Figure 3

Induction of Kip1 mRNA expression in 5L cells by TCDD. (A) Kip1 mRNA levels were determined by Northern blot analysis in 5L cells after 4 hr exposure to TCDD with or without 30 min of pretreatment with the translational inhibitor cycloheximide (50 μg/ml). Cycloheximide treatment was efficient because p27^Kip1 protein accumulation was prevented (third panel) whereas levels of a noninducible protein (p38^HOG1 kinase) remained unchanged in the same extracts. (B,C) Kip1 mRNA stability in control or TCDD-treated 5L cells was analyzed after induction for 4 hr and disruption of further transcription by actinomycin D (5 μg/ml). The decay of Kip1 mRNA was followed by Northern blot analysis and PhosphorImager quantification in comparison to GAPDH mRNA. mRNA half-life times were determined by regression analysis from the presentation in C. (□) −TCDD; (█) +TCDD. (D) The transcriptional rate of the Kip1 gene was analyzed by a nuclear run-off analysis (Greenberg and Ziff 1984) with nuclei prepared from 5L cells that had been treated for 4 hr or 24 hr with 1 nm TCDD or the DMSO solvent. A representative result (24 hr) is shown. (E) Kip1 promoter reporter gene constructs (Kwon et al. 1996) comprising either 1609, 1382, or 42 bp upstream of the transcriptional start site were stably integrated into 5L cells. Also a Kip1 promoter fragment comprising base pairs −1609 to −1312 in front of a TK₈₁-promoter–luciferase reporter gene (Nordeen 1988) was stably transfected. Inducibility in cell pools (average values of triplicate determinations ± s.d.) was calculated as the ratio of reporter gene activity from cells that had been treated for 24 hr with 1 nm TCDD or the solvent, respectively. (F) Kip1 mRNA expression and inducibility by TCDD were tested in 5L cells that had been serum starved for 24 hr. Cells were treated for additional 4 hr in serum-free medium with 1 nm TCDD or the DMSO solvent prior to Northern blot analysis. Fractions of cells in the S–G₂/M segment of the cell cycle were determined from parallel cultures by flow cytometry. (N.D.) Not determined.

Figure 3

Induction of Kip1 mRNA expression in 5L cells by TCDD. (A) Kip1 mRNA levels were determined by Northern blot analysis in 5L cells after 4 hr exposure to TCDD with or without 30 min of pretreatment with the translational inhibitor cycloheximide (50 μg/ml). Cycloheximide treatment was efficient because p27^Kip1 protein accumulation was prevented (third panel) whereas levels of a noninducible protein (p38^HOG1 kinase) remained unchanged in the same extracts. (B,C) Kip1 mRNA stability in control or TCDD-treated 5L cells was analyzed after induction for 4 hr and disruption of further transcription by actinomycin D (5 μg/ml). The decay of Kip1 mRNA was followed by Northern blot analysis and PhosphorImager quantification in comparison to GAPDH mRNA. mRNA half-life times were determined by regression analysis from the presentation in C. (□) −TCDD; (█) +TCDD. (D) The transcriptional rate of the Kip1 gene was analyzed by a nuclear run-off analysis (Greenberg and Ziff 1984) with nuclei prepared from 5L cells that had been treated for 4 hr or 24 hr with 1 nm TCDD or the DMSO solvent. A representative result (24 hr) is shown. (E) Kip1 promoter reporter gene constructs (Kwon et al. 1996) comprising either 1609, 1382, or 42 bp upstream of the transcriptional start site were stably integrated into 5L cells. Also a Kip1 promoter fragment comprising base pairs −1609 to −1312 in front of a TK₈₁-promoter–luciferase reporter gene (Nordeen 1988) was stably transfected. Inducibility in cell pools (average values of triplicate determinations ± s.d.) was calculated as the ratio of reporter gene activity from cells that had been treated for 24 hr with 1 nm TCDD or the solvent, respectively. (F) Kip1 mRNA expression and inducibility by TCDD were tested in 5L cells that had been serum starved for 24 hr. Cells were treated for additional 4 hr in serum-free medium with 1 nm TCDD or the DMSO solvent prior to Northern blot analysis. Fractions of cells in the S–G₂/M segment of the cell cycle were determined from parallel cultures by flow cytometry. (N.D.) Not determined.

Figure 4

p27^Kip1 protein half life and rate of synthesis. (A) Protein half life of p27^Kip1 in 5L cells was analyzed by protein pulse labeling with [³⁵S]methionine for 3 hr in the presence of 1 nm TCDD or DMSO solvent followed by a chase for 0, 3, or 6 hr with nonlabeled methionine. The autoradiogram of immune precipitated p27^Kip1 is shown (solid arrow) together with a nonspecifically precipitated protein (open arrow). (B) Band intensities in A were quantitated from scanned X-ray films using the calibrated NIH image 1.61 software. (□) −TCDD; (█) +TCDD. (C) The rate of p27^Kip1 protein synthesis was determined by [³⁵S]methionine pulse labeling (1 hr) followed by immune precipitation of p27^Kip1. Cells had been pretreated for 2 hr 45 min with actinomycin D (5 μg/ml), 2 hr 30 min TCDD (1 nm), both, or the solvent (DMSO) only. One out of two similar experiments, each, are shown.

Figure 4

p27^Kip1 protein half life and rate of synthesis. (A) Protein half life of p27^Kip1 in 5L cells was analyzed by protein pulse labeling with [³⁵S]methionine for 3 hr in the presence of 1 nm TCDD or DMSO solvent followed by a chase for 0, 3, or 6 hr with nonlabeled methionine. The autoradiogram of immune precipitated p27^Kip1 is shown (solid arrow) together with a nonspecifically precipitated protein (open arrow). (B) Band intensities in A were quantitated from scanned X-ray films using the calibrated NIH image 1.61 software. (□) −TCDD; (█) +TCDD. (C) The rate of p27^Kip1 protein synthesis was determined by [³⁵S]methionine pulse labeling (1 hr) followed by immune precipitation of p27^Kip1. Cells had been pretreated for 2 hr 45 min with actinomycin D (5 μg/ml), 2 hr 30 min TCDD (1 nm), both, or the solvent (DMSO) only. One out of two similar experiments, each, are shown.

Figure 4

p27^Kip1 protein half life and rate of synthesis. (A) Protein half life of p27^Kip1 in 5L cells was analyzed by protein pulse labeling with [³⁵S]methionine for 3 hr in the presence of 1 nm TCDD or DMSO solvent followed by a chase for 0, 3, or 6 hr with nonlabeled methionine. The autoradiogram of immune precipitated p27^Kip1 is shown (solid arrow) together with a nonspecifically precipitated protein (open arrow). (B) Band intensities in A were quantitated from scanned X-ray films using the calibrated NIH image 1.61 software. (□) −TCDD; (█) +TCDD. (C) The rate of p27^Kip1 protein synthesis was determined by [³⁵S]methionine pulse labeling (1 hr) followed by immune precipitation of p27^Kip1. Cells had been pretreated for 2 hr 45 min with actinomycin D (5 μg/ml), 2 hr 30 min TCDD (1 nm), both, or the solvent (DMSO) only. One out of two similar experiments, each, are shown.

Figure 5

Time course of Kip1 mRNA and protein induction by TCDD in 5L cells. Expression of Kip1 mRNA and protein in 5L cells after the indicated times of TCDD treatment was determined by Northern blot analysis of poly(A)⁺ RNA (top) or Western blot analysis of total cell extracts (bottom), respectively.

Figure 6

Expression of Kip1 antisense RNA impairs TCDD-induced inhibition of 5L cell proliferation. Proliferation rates in the presence or absence of TCDD were determined in 5L cells that had been transiently cotransfected with expression vectors for a Kip1 antisense RNA and the GFP (anti-Kip1) or the empty expression vector and GFP (control). Green fluorescing cells were collected by FACS and analyzed by either Western blotting for p27^Kip1 levels (A) or the percentage of cells that had incorporated BrdU in the presence (solid bars) or absence (open bars) of TCDD (B) (means ± s.d. from three independent experiments in which BrdU staining was evaluated without prior knowledge of sample identity). Significance of differences was tested by Student’s t-test.

Figure 6

Expression of Kip1 antisense RNA impairs TCDD-induced inhibition of 5L cell proliferation. Proliferation rates in the presence or absence of TCDD were determined in 5L cells that had been transiently cotransfected with expression vectors for a Kip1 antisense RNA and the GFP (anti-Kip1) or the empty expression vector and GFP (control). Green fluorescing cells were collected by FACS and analyzed by either Western blotting for p27^Kip1 levels (A) or the percentage of cells that had incorporated BrdU in the presence (solid bars) or absence (open bars) of TCDD (B) (means ± s.d. from three independent experiments in which BrdU staining was evaluated without prior knowledge of sample identity). Significance of differences was tested by Student’s t-test.

Figure 7

TCDD-induced p27^Kip1 protein and cell cycle delay in FTOCs. Fetal thymus glands were cultured for two days in the presence of TCDD or the DMSO solvent before preparation of thymocytes. (A) p27^Kip1 protein in thymocytes was determined by Western blot analysis and even loading was confirmed by reprobing with an Erk1 antibody. (B) Incorporation of [3^H]thymidine was quantitated per amount of genomic thymocyte DNA after addition of [3^H]thymidine for the last 12 hr of culture. (C) The cell cycle distribution of total thymocytes after 2 days of culture was determined by flow cytometry after DNA staining with H33258. (D) Thymocyte subpopulations after 2 days of culture were analyzed by cell surface staining and flow cytometry. One representative out of three experiments is shown; the numbers in B and C are means ± s.d. from three independent experiments (P < 0.01).

Figure 8

FTOC from wild-type or Kip1 mutant mice. FTOCs were prepared from wild-type (WT), Kip1 mutant (Δ51/Δ51), or heterozygous littermate embryos. A representative PCR-based genotyping for Kip1 is shown in A. Genotyping for AhR alleles (B) was performed by Western blot analysis of the encoded proteins. Liver cytosolic extracts from C57/Bl6 (AhR^b1 allele) and 129/Sv (AhR^dallele) mice were loaded as reference together with extracts from AhR^b/b or AhR^b/d embryos. (Solid arrowhead) The size of AhR^b1 (95 kD) and a putative degradation product (82 kD) that is specific for the C57Bl6 mouse; (open arrowhead) AhR^d; (⋄) a prominent unspecific band. (C) The effect of TCDD on cell cycle distribution depending on the Kip1 genotype in FTOCs from embryos that carry at least one allele coding for a high-affinity AhR (b1 allele). The fraction of cells in mid-S–G₂/M in each of the solvent-treated control thymic lobes was set to 100% and the pair-matched TCDD-treated lobe was evaluated relative to that. Data from six litters were grouped according to genotyping of the embryos (Means ± s.e.: n_WT = 10; n_{Kip1 WT/Δ51} = 4; n_{Kip1 Δ51/Δ51} = 5). (Solid bars) +TCDD; (open bars) −TCDD. TCDD effects in each group and differences between groups were statistically evaluated by Student’s t-test. (D) Thymocyte recovery after 7 days of FTOCs in the absence or presence of TCDD was determined by hemocytometer counting. For each pair of thymic lobes isolated from an individual embryo the cell number recovered from the solvent (0.1% DMSO) treated lobe was set to 100% and the recovery from the TCDD-treated lobe (1 nm) was calculated as relative value. Data from three litters were grouped according to Kip1 genotyping and confirmation of the presence of at least one high-affinity AhR allele. Values are means ± s.e. (n_wt = 4, n_het = 5, n_{Kip1 Δ51/Δ51} = 6) and significance levels were calculated by Student’s t-test.