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The putative effects of UCN-01 on protein kinase C and/or PDK1/AKT are dispensable for the induction of p21. A, HaCaT cells were exposed to increasing concentrations of UCN-01 (10 – 300 n M ) or increasing concentrations of
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The small molecule UCN-01 is a cyclin-dependent kinase (CDK) modulator shown to have antiproliferative effects against several in vitro and in vivo cancer models currently being tested in human clinical trials. Although UCN-01 may inhibit several serine-threonine kinases, the exact mechanism by which it promotes cell cycle arrest is still unclear....
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... in Fig. 4, UCN-01 significantly enhanced the transcriptional activity of the p21 full-length 2.4 kb promoter (p21P-luc) by ϳ 5-fold. Similar activation ( ϳ 6-fold) was observed with Ras V12 (positive control). Moreover, we transfected HaCaT cell lines (a keratinocyte cell line with deficient p53 function; Ref. 31) with the p21P ⌬ p53-luc reporter, a p21 waf1/cip1 construct lacking 250 bases from the 5 Ј end corresponding to the p53 consensus DNA binding site. Again, UCN-01 activates this construct ( ϳ 6-fold) despite the lack of the p53 consensus site, indicating that neither the presence of p53 binding sites in the p21 waf1/cip1 promoter nor the presence of functional p53 protein was required for UCN-01 to activate the p21 waf1/cip1 promoter. To determine the minimal promoter region required for the transcriptional activation of p21 waf1/cip1 by UCN-01, additional promoter deletion constructs were tested. We initially used the p21PSma-luc construct (26, 30), which contains the p21 waf1/cip1 promoter sequences from base Ϫ 111 through the transcriptional initiation site. Of note, this construct represents the minimal promoter region activated by Ras (26, 30). As shown in Fig. 4, UCN-01 significantly activates this minimal promoter ( ϳ 7-fold), similar to Ras V12. Moreover, when HaCaT cells were transfected with full-length p21 waf1/cip1 construct lacking the Ras minimal promoter region p21PSma ⌬ 1-luc the induction was lost for both Ras V12 and UCN-01. Thus, the minimal p21 waf1/cip1 promoter region for UCN-01 is similar to that of Ras, and it seems to be represented by a region in the p21 waf1/cip1 promoter proximal to the Sma I site at Ϫ 111 (p21PSma-luc). To test whether the p21 waf1/cip1 transcriptional activation by UCN-01 occurs in other cell types, we transfected pSMA-luc into HCT116 isogenic cell lines (wild-type or p53 null cells). Again, UCN-01 activated pSMA-luc in both cell lines (data not shown), demonstrating that the induction of the p21 minimal promoter by UCN-01 occurs in other cell types and is also independent of p53 function. Require PKC nor PDK1/AKT Pathways. UCN-01 is a known inhibitor of several serine-threonine kinases, including PKC and PDK1 (14, 32, 33). To test whether the p21 transcriptional effects of UCN-01 might be related to PKC modulation, we assessed whether the putative effects of UCN-01 on PKC function occur at times and concentrations relevant for the p21 induction induced by this agent. To this end, we assessed the phosphorylation of adducin, a known substrate of PKC (16, 34). As demonstrated in Fig. 5 A, the phosphorylation of adducin was unaltered even at the highest UCN-01 concentrations tested. Thus, the putative effects of UCN-01 in PKC activity appear not to be relevant for p21 transcription, as demonstrated previously with respect to apoptosis and cell cycle arrest (15, 17). Sato et al. (33) demonstrated recently that UCN-01 inhibits PDK1, a serine-threonine kinase responsible for the activation of AKT (35, 36). Of note, fully active AKT requires the phosphorylation of 2 residues, threonine 308 (site phosphorylated by PDK1) and serine 473 (site phosphorylated by an undefined kinase; Refs. 35, 36). To assess whether UCN-01 activates p21 transcription by modulation of AKT signaling, we tested whether UCN-01 promotes the loss in AKT phosphorylation in HaCaT cell lines. As shown in Fig. 5 A, although UCN-01 induced p21 at concentrations Ͼ 30 n M (as shown before), the effects of UCN-01 on dephosphorylation of AKT on site S473 (PDK- independent site) occurred only at concentrations Ͼ 300 n M . Moreover, the PDK1-dependent site, threonine 308, was unchanged at the times and concentrations required for p21 induction by UCN-01. In contrast, the small molecule PI3k inhibitor LY294002 dephosphoryl- ated AKT at both residues (threonine 308 and serine 473) while having minimal effects on p21 expression (Fig. 5 A ). Similar results were observed with another chemically unrelated PI3k inhibitor, wortmannin (data not shown). Furthermore, coexposure of HaCaT cells to UCN-01 in combination with either LY294002 or wortmannin showed unaltered induction of p21 (data not shown). These results suggest that induction of p21 by UCN-01 is not related to the capacity of UCN-01 to inhibit PI3k/AKT pathway. To additionally exclude the possibility that the effects of UCN-01 in the PDK1/AKT pathway might be responsible for p21 up-regulation, we expressed a constitutively active form of Akt (Myr-Akt) and a kinase-inactive form of Akt (T308A and S473A) in HaCaT cells using adenoviral delivery. Upon infection with increasing multiplicity of infection of the AKT AdVs, a dose-dependent increase in the expression of the corresponding proteins was detected, as verified by Western blot using an antibody against total AKT (Fig. 5 B ). Then, HaCaT cells infected with myr-AKT adenovirus were exposed to UCN-01. Consistent with Fig. 3, p21 was significantly increased in HaCaT cells exposed to UCN-01 (Fig. 5 B, Lane 6 ). Increasing multiplicity of infection of activated AKT failed to prevent this up- regulation (Fig. 5 B, Lanes 7–10 ). Moreover, increasing multiplicity of infection with kinase-deficient AKT failed to increase p21, strongly suggesting that the effects of UCN-01 in p21 expression appear not to be related to the effects on the PDK1/AKT pathway reported previously. Activation of the MEK/ERK Pathway. Because the transcriptional up-regulation of p21 by UCN-01 is p53 independent and the minimal promoter region is similar to the one required for the proto-oncogene Ras to activate the p21 promoter, we tested whether UCN-01 could modulate signal pathways downstream to Ras. Ras modulates transcriptional events by activating the mitogen-activated protein kinase (MAPK) kinase kinase, Raf (37 – 40). In turn, Raf activates MEK and this, in turn, phosphorylates and activates the ERK1 and ERK2, leading to the phosphorylation and activation of several transcriptional factors. To test whether UCN-01 activates the MEK/ERK pathway, we exposed serum-starved HaCaT cells to increasing concentrations of UCN-01 for 60 min or for increasing time periods followed by in vitro kinase reactions. UCN-01 at concentrations Ն 100 n M significantly activated ERK kinase activity (Fig. 6 A ). Similar results were obtained when protein lysates were immunoblotted with a phospho-specific ERK1/2 antibody (data not shown). Time course analysis revealed that 30 min of 100 n M UCN-01 exposure were sufficient to activate the MEK/ERK pathway (Fig. 6 A, right ). Thus, MAPK activation occurs at similar concentrations ( Ն 100 n M ) required for p21 up-regulation. Moreover, as demonstrated in Fig. 6 A, the activation of MAPK temporally precedes the increase in p21 mRNA (3 – 6 h; Fig. 3 C ). To confirm the involvement of the MEK/ERK pathway in UCN-01-induced p21 transcriptional up-regulation, we used two structurally unrelated chemical inhibitors of the MAPK kinase, MEK (PD98059 and U0126), at concentrations that were specific for MEK inhibition (41, 42). HaCaT cells either exposed to UCN-01 (Fig. 6 B, Lane 2 ) or transfected with Ras (Fig. 6 B, Lane 5 ) demonstrated an increase in p21 promoter activity, as previously shown in Fig. 4. However, cotransfection with dominant-negative MEK (MEKAA) or preincubation with the MEK inhibitor PD98059 blunted the activation of the p21 promoter by either UCN-01 or Ras. Similar results were obtained when p21 expression was determined by Northern blot studies (Fig. 6 C ). Activation of the MEK/ERK Pathway. To test whether MAPK was also required for the induction of p21 at the protein level, serum- starved HaCaT cells treated with UCN-01 were preincubated with the chemical MEK inhibitor PD98059. Again, the loss in MEK activity induced by PD98059, as measured by loss in ERK phosphorylation, blunted the p21 protein induction induced by UCN-01 (Fig. 7 A ). Moreover, we assessed whether the activation of MEK occurs also in serum containing exponentially growing conditions. To this end, subconfluent HaCaT cells treated with UCN-01 were preincubated with another structurally unrelated MEK inhibitor, UO126. As clearly demonstrated in Fig. 7 B, the induction of p21 by UCN-01 was associated with activation of MEK, as measured by antiserum that only recognizes phosphorylated MEK. Moreover, preincubation with the MEK inhibitor blunted the induction of p21 by UCN-01. Together, UCN-01 induces the activation of MEK/ERK pathways in both serum-free and serum-containing conditions, and this activation is required for the up-regulation of p21. Next, we performed immunofluorescence studies to assess whether the p21 accumulation induced by UCN-01 was nuclear or cytoplasmic and to test whether MEK blockade prevented not only the induction but also the redistribution of p21. As clearly demonstrated in Fig. 7 B, UCN-01 significantly increased p21 expression, similarly to what was observed previously at the Western blot level (Fig. 3). Moreover, the increase in p21 was exclusively nuclear as the immunostaining colo- calized with 4 Ј ,6-diamidino-2-phenylindole, a known nuclear fluores- cence stain, without evidence of redistribution to the cytoplasmic compartment (see Fig. 7 C, inset ). Furthermore, preincubation with the MEK inhibitor PD98059 significantly diminished the total expression of nuclear p21. Thus, induction of p21 by UCN-01 appears to be almost exclusively nuclear, and its expression requires the activation of MEK/ERK pathway. In summary, UCN-01 promotes cell cycle arrest through an increase in p21 protein expression. This increase is p53 independent and is due to transcriptional activation of the p21 waf1/cip1 promoter. The minimal promoter region of p21 is similar to the one activated by Ras, and p21 transcriptional activation by UCN-01 requires the activation of the MEK/MAPK pathway. This finding contributes to the under- standing of the mechanism of cell cycle arrest by this compound and may help also in ...
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... pulse-chase analysis in the presence of actinomycin D and measured p21 mRNA by Northern blot studies. We observed that p21 mRNA expression decreased to a similar extent in both vehicle and UCN-01-treated cells up to 6 h after actinomycin D (85% and 82%, respectively). These experiments demonstrate that p21 accumulation promoted by UCN-01 is not explained by increased in p21 mRNA half-life. Instead, it appears to be due to transcriptional effects. quired for Ras to Activate the p21 Promoter. To confirm that the increase in p21 waf1/cip1 mRNA induced by UCN-01 was due to transcriptional activation of the p21 waf1/cip1 promoter, we transiently transfected HaCaT cells with several p21 waf1/cip1 promoter-driven luciferase reporter plasmids (26, 30). Twelve h after transfection, HaCaT cells were exposed to 100 n M UCN-01 for 18 h, and luciferase activity was measured as described in “ Materials and Methods. ” As a positive control, we transiently transfected HaCaT cells with activated Ras (Ras V12), a known transcriptional activator of the p21 waf1/cip1 promoter (26). As shown in Fig. 4, UCN-01 significantly enhanced the transcriptional activity of the p21 full-length 2.4 kb promoter (p21P-luc) by ϳ 5-fold. Similar activation ( ϳ 6-fold) was observed with Ras V12 (positive control). Moreover, we transfected HaCaT cell lines (a keratinocyte cell line with deficient p53 function; Ref. 31) with the p21P ⌬ p53-luc reporter, a p21 waf1/cip1 construct lacking 250 bases from the 5 Ј end corresponding to the p53 consensus DNA binding site. Again, UCN-01 activates this construct ( ϳ 6-fold) despite the lack of the p53 consensus site, indicating that neither the presence of p53 binding sites in the p21 waf1/cip1 promoter nor the presence of functional p53 protein was required for UCN-01 to activate the p21 waf1/cip1 promoter. To determine the minimal promoter region required for the transcriptional activation of p21 waf1/cip1 by UCN-01, additional promoter deletion constructs were tested. We initially used the p21PSma-luc construct (26, 30), which contains the p21 waf1/cip1 promoter sequences from base Ϫ 111 through the transcriptional initiation site. Of note, this construct represents the minimal promoter region activated by Ras (26, 30). As shown in Fig. 4, UCN-01 significantly activates this minimal promoter ( ϳ 7-fold), similar to Ras V12. Moreover, when HaCaT cells were transfected with full-length p21 waf1/cip1 construct lacking the Ras minimal promoter region p21PSma ⌬ 1-luc the induction was lost for both Ras V12 and UCN-01. Thus, the minimal p21 waf1/cip1 promoter region for UCN-01 is similar to that of Ras, and it seems to be represented by a region in the p21 waf1/cip1 promoter proximal to the Sma I site at Ϫ 111 (p21PSma-luc). To test whether the p21 waf1/cip1 transcriptional activation by UCN-01 occurs in other cell types, we transfected pSMA-luc into HCT116 isogenic cell lines (wild-type or p53 null cells). Again, UCN-01 activated pSMA-luc in both cell lines (data not shown), demonstrating that the induction of the p21 minimal promoter by UCN-01 occurs in other cell types and is also independent of p53 function. Require PKC nor PDK1/AKT Pathways. UCN-01 is a known inhibitor of several serine-threonine kinases, including PKC and PDK1 (14, 32, 33). To test whether the p21 transcriptional effects of UCN-01 might be related to PKC modulation, we assessed whether the putative effects of UCN-01 on PKC function occur at times and concentrations relevant for the p21 induction induced by this agent. To this end, we assessed the phosphorylation of adducin, a known substrate of PKC (16, 34). As demonstrated in Fig. 5 A, the phosphorylation of adducin was unaltered even at the highest UCN-01 concentrations tested. Thus, the putative effects of UCN-01 in PKC activity appear not to be relevant for p21 transcription, as demonstrated previously with respect to apoptosis and cell cycle arrest (15, 17). Sato et al. (33) demonstrated recently that UCN-01 inhibits PDK1, a serine-threonine kinase responsible for the activation of AKT (35, 36). Of note, fully active AKT requires the phosphorylation of 2 residues, threonine 308 (site phosphorylated by PDK1) and serine 473 (site phosphorylated by an undefined kinase; Refs. 35, 36). To assess whether UCN-01 activates p21 transcription by modulation of AKT signaling, we tested whether UCN-01 promotes the loss in AKT phosphorylation in HaCaT cell lines. As shown in Fig. 5 A, although UCN-01 induced p21 at concentrations Ͼ 30 n M (as shown before), the effects of UCN-01 on dephosphorylation of AKT on site S473 (PDK- independent site) occurred only at concentrations Ͼ 300 n M . Moreover, the PDK1-dependent site, threonine 308, was unchanged at the times and concentrations required for p21 induction by UCN-01. In contrast, the small molecule PI3k inhibitor LY294002 dephosphoryl- ated AKT at both residues (threonine 308 and serine 473) while having minimal effects on p21 expression (Fig. 5 A ). Similar results were observed with another chemically unrelated PI3k inhibitor, wortmannin (data not shown). Furthermore, coexposure of HaCaT cells to UCN-01 in combination with either LY294002 or wortmannin showed unaltered induction of p21 (data not shown). These results suggest that induction of p21 by UCN-01 is not related to the capacity of UCN-01 to inhibit PI3k/AKT pathway. To additionally exclude the possibility that the effects of UCN-01 in the PDK1/AKT pathway might be responsible for p21 up-regulation, we expressed a constitutively active form of Akt (Myr-Akt) and a kinase-inactive form of Akt (T308A and S473A) in HaCaT cells using adenoviral delivery. Upon infection with increasing multiplicity of infection of the AKT AdVs, a dose-dependent increase in the expression of the corresponding proteins was detected, as verified by Western blot using an antibody against total AKT (Fig. 5 B ). Then, HaCaT cells infected with myr-AKT adenovirus were exposed to UCN-01. Consistent with Fig. 3, p21 was significantly increased in HaCaT cells exposed to UCN-01 (Fig. 5 B, Lane 6 ). Increasing multiplicity of infection of activated AKT failed to prevent this up- regulation (Fig. 5 B, Lanes 7–10 ). Moreover, increasing multiplicity of infection with kinase-deficient AKT failed to increase p21, strongly suggesting that the effects of UCN-01 in p21 expression appear not to be related to the effects on the PDK1/AKT pathway reported previously. Activation of the MEK/ERK Pathway. Because the transcriptional up-regulation of p21 by UCN-01 is p53 independent and the minimal promoter region is similar to the one required for the proto-oncogene Ras to activate the p21 promoter, we tested whether UCN-01 could modulate signal pathways downstream to Ras. Ras modulates transcriptional events by activating the mitogen-activated protein kinase (MAPK) kinase kinase, Raf (37 – 40). In turn, Raf activates MEK and this, in turn, phosphorylates and activates the ERK1 and ERK2, leading to the phosphorylation and activation of several transcriptional factors. To test whether UCN-01 activates the MEK/ERK pathway, we exposed serum-starved HaCaT cells to increasing concentrations of UCN-01 for 60 min or for increasing time periods followed by in vitro kinase reactions. UCN-01 at concentrations Ն 100 n M significantly activated ERK kinase activity (Fig. 6 A ). Similar results were obtained when protein lysates were immunoblotted with a phospho-specific ERK1/2 antibody (data not shown). Time course analysis revealed that 30 min of 100 n M UCN-01 exposure were sufficient to activate the MEK/ERK pathway (Fig. 6 A, right ). Thus, MAPK activation occurs at similar concentrations ( Ն 100 n M ) required for p21 up-regulation. Moreover, as demonstrated in Fig. 6 A, the activation of MAPK temporally precedes the increase in p21 mRNA (3 – 6 h; Fig. 3 C ). To confirm the involvement of the MEK/ERK pathway in UCN-01-induced p21 transcriptional up-regulation, we used two structurally unrelated chemical inhibitors of the MAPK kinase, MEK (PD98059 and U0126), at concentrations that were specific for MEK inhibition (41, 42). HaCaT cells either exposed to UCN-01 (Fig. 6 B, Lane 2 ) or transfected with Ras (Fig. 6 B, Lane 5 ) demonstrated an increase in p21 promoter activity, as previously shown in Fig. 4. However, cotransfection with dominant-negative MEK (MEKAA) or preincubation with the MEK inhibitor PD98059 blunted the activation of the p21 promoter by either UCN-01 or Ras. Similar results were obtained when p21 expression was determined by Northern blot studies (Fig. 6 C ). Activation of the MEK/ERK Pathway. To test whether MAPK was also required for the induction of p21 at the protein level, serum- starved HaCaT cells treated with UCN-01 were preincubated with the chemical MEK inhibitor PD98059. Again, the loss in MEK activity induced by PD98059, as measured by loss in ERK phosphorylation, blunted the p21 protein induction induced by UCN-01 (Fig. 7 A ). Moreover, we assessed whether the activation of MEK occurs also in serum containing exponentially growing conditions. To this end, subconfluent HaCaT cells treated with UCN-01 were preincubated with another structurally unrelated MEK inhibitor, UO126. As clearly demonstrated in Fig. 7 B, the induction of p21 by UCN-01 was associated with activation of MEK, as measured by antiserum that only recognizes phosphorylated MEK. Moreover, preincubation with the MEK inhibitor blunted the induction of p21 by UCN-01. Together, UCN-01 induces the activation of MEK/ERK pathways in both serum-free and serum-containing conditions, and this activation is required for the up-regulation of p21. Next, we performed immunofluorescence studies to assess whether the p21 accumulation induced by UCN-01 was nuclear or cytoplasmic and to test whether MEK blockade prevented not only the induction but also the redistribution of p21. As clearly demonstrated in Fig. 7 B, UCN-01 significantly increased p21 ...
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... ” As a positive control, we transiently transfected HaCaT cells with activated Ras (Ras V12), a known transcriptional activator of the p21 waf1/cip1 promoter (26). As shown in Fig. 4, UCN-01 significantly enhanced the transcriptional activity of the p21 full-length 2.4 kb promoter (p21P-luc) by ϳ 5-fold. Similar activation ( ϳ 6-fold) was observed with Ras V12 (positive control). Moreover, we transfected HaCaT cell lines (a keratinocyte cell line with deficient p53 function; Ref. 31) with the p21P ⌬ p53-luc reporter, a p21 waf1/cip1 construct lacking 250 bases from the 5 Ј end corresponding to the p53 consensus DNA binding site. Again, UCN-01 activates this construct ( ϳ 6-fold) despite the lack of the p53 consensus site, indicating that neither the presence of p53 binding sites in the p21 waf1/cip1 promoter nor the presence of functional p53 protein was required for UCN-01 to activate the p21 waf1/cip1 promoter. To determine the minimal promoter region required for the transcriptional activation of p21 waf1/cip1 by UCN-01, additional promoter deletion constructs were tested. We initially used the p21PSma-luc construct (26, 30), which contains the p21 waf1/cip1 promoter sequences from base Ϫ 111 through the transcriptional initiation site. Of note, this construct represents the minimal promoter region activated by Ras (26, 30). As shown in Fig. 4, UCN-01 significantly activates this minimal promoter ( ϳ 7-fold), similar to Ras V12. Moreover, when HaCaT cells were transfected with full-length p21 waf1/cip1 construct lacking the Ras minimal promoter region p21PSma ⌬ 1-luc the induction was lost for both Ras V12 and UCN-01. Thus, the minimal p21 waf1/cip1 promoter region for UCN-01 is similar to that of Ras, and it seems to be represented by a region in the p21 waf1/cip1 promoter proximal to the Sma I site at Ϫ 111 (p21PSma-luc). To test whether the p21 waf1/cip1 transcriptional activation by UCN-01 occurs in other cell types, we transfected pSMA-luc into HCT116 isogenic cell lines (wild-type or p53 null cells). Again, UCN-01 activated pSMA-luc in both cell lines (data not shown), demonstrating that the induction of the p21 minimal promoter by UCN-01 occurs in other cell types and is also independent of p53 function. Require PKC nor PDK1/AKT Pathways. UCN-01 is a known inhibitor of several serine-threonine kinases, including PKC and PDK1 (14, 32, 33). To test whether the p21 transcriptional effects of UCN-01 might be related to PKC modulation, we assessed whether the putative effects of UCN-01 on PKC function occur at times and concentrations relevant for the p21 induction induced by this agent. To this end, we assessed the phosphorylation of adducin, a known substrate of PKC (16, 34). As demonstrated in Fig. 5 A, the phosphorylation of adducin was unaltered even at the highest UCN-01 concentrations tested. Thus, the putative effects of UCN-01 in PKC activity appear not to be relevant for p21 transcription, as demonstrated previously with respect to apoptosis and cell cycle arrest (15, 17). Sato et al. (33) demonstrated recently that UCN-01 inhibits PDK1, a serine-threonine kinase responsible for the activation of AKT (35, 36). Of note, fully active AKT requires the phosphorylation of 2 residues, threonine 308 (site phosphorylated by PDK1) and serine 473 (site phosphorylated by an undefined kinase; Refs. 35, 36). To assess whether UCN-01 activates p21 transcription by modulation of AKT signaling, we tested whether UCN-01 promotes the loss in AKT phosphorylation in HaCaT cell lines. As shown in Fig. 5 A, although UCN-01 induced p21 at concentrations Ͼ 30 n M (as shown before), the effects of UCN-01 on dephosphorylation of AKT on site S473 (PDK- independent site) occurred only at concentrations Ͼ 300 n M . Moreover, the PDK1-dependent site, threonine 308, was unchanged at the times and concentrations required for p21 induction by UCN-01. In contrast, the small molecule PI3k inhibitor LY294002 dephosphoryl- ated AKT at both residues (threonine 308 and serine 473) while having minimal effects on p21 expression (Fig. 5 A ). Similar results were observed with another chemically unrelated PI3k inhibitor, wortmannin (data not shown). Furthermore, coexposure of HaCaT cells to UCN-01 in combination with either LY294002 or wortmannin showed unaltered induction of p21 (data not shown). These results suggest that induction of p21 by UCN-01 is not related to the capacity of UCN-01 to inhibit PI3k/AKT pathway. To additionally exclude the possibility that the effects of UCN-01 in the PDK1/AKT pathway might be responsible for p21 up-regulation, we expressed a constitutively active form of Akt (Myr-Akt) and a kinase-inactive form of Akt (T308A and S473A) in HaCaT cells using adenoviral delivery. Upon infection with increasing multiplicity of infection of the AKT AdVs, a dose-dependent increase in the expression of the corresponding proteins was detected, as verified by Western blot using an antibody against total AKT (Fig. 5 B ). Then, HaCaT cells infected with myr-AKT adenovirus were exposed to UCN-01. Consistent with Fig. 3, p21 was significantly increased in HaCaT cells exposed to UCN-01 (Fig. 5 B, Lane 6 ). Increasing multiplicity of infection of activated AKT failed to prevent this up- regulation (Fig. 5 B, Lanes 7–10 ). Moreover, increasing multiplicity of infection with kinase-deficient AKT failed to increase p21, strongly suggesting that the effects of UCN-01 in p21 expression appear not to be related to the effects on the PDK1/AKT pathway reported previously. Activation of the MEK/ERK Pathway. Because the transcriptional up-regulation of p21 by UCN-01 is p53 independent and the minimal promoter region is similar to the one required for the proto-oncogene Ras to activate the p21 promoter, we tested whether UCN-01 could modulate signal pathways downstream to Ras. Ras modulates transcriptional events by activating the mitogen-activated protein kinase (MAPK) kinase kinase, Raf (37 – 40). In turn, Raf activates MEK and this, in turn, phosphorylates and activates the ERK1 and ERK2, leading to the phosphorylation and activation of several transcriptional factors. To test whether UCN-01 activates the MEK/ERK pathway, we exposed serum-starved HaCaT cells to increasing concentrations of UCN-01 for 60 min or for increasing time periods followed by in vitro kinase reactions. UCN-01 at concentrations Ն 100 n M significantly activated ERK kinase activity (Fig. 6 A ). Similar results were obtained when protein lysates were immunoblotted with a phospho-specific ERK1/2 antibody (data not shown). Time course analysis revealed that 30 min of 100 n M UCN-01 exposure were sufficient to activate the MEK/ERK pathway (Fig. 6 A, right ). Thus, MAPK activation occurs at similar concentrations ( Ն 100 n M ) required for p21 up-regulation. Moreover, as demonstrated in Fig. 6 A, the activation of MAPK temporally precedes the increase in p21 mRNA (3 – 6 h; Fig. 3 C ). To confirm the involvement of the MEK/ERK pathway in UCN-01-induced p21 transcriptional up-regulation, we used two structurally unrelated chemical inhibitors of the MAPK kinase, MEK (PD98059 and U0126), at concentrations that were specific for MEK inhibition (41, 42). HaCaT cells either exposed to UCN-01 (Fig. 6 B, Lane 2 ) or transfected with Ras (Fig. 6 B, Lane 5 ) demonstrated an increase in p21 promoter activity, as previously shown in Fig. 4. However, cotransfection with dominant-negative MEK (MEKAA) or preincubation with the MEK inhibitor PD98059 blunted the activation of the p21 promoter by either UCN-01 or Ras. Similar results were obtained when p21 expression was determined by Northern blot studies (Fig. 6 C ). Activation of the MEK/ERK Pathway. To test whether MAPK was also required for the induction of p21 at the protein level, serum- starved HaCaT cells treated with UCN-01 were preincubated with the chemical MEK inhibitor PD98059. Again, the loss in MEK activity induced by PD98059, as measured by loss in ERK phosphorylation, blunted the p21 protein induction induced by UCN-01 (Fig. 7 A ). Moreover, we assessed whether the activation of MEK occurs also in serum containing exponentially growing conditions. To this end, subconfluent HaCaT cells treated with UCN-01 were preincubated with another structurally unrelated MEK inhibitor, UO126. As clearly demonstrated in Fig. 7 B, the induction of p21 by UCN-01 was associated with activation of MEK, as measured by antiserum that only recognizes phosphorylated MEK. Moreover, preincubation with the MEK inhibitor blunted the induction of p21 by UCN-01. Together, UCN-01 induces the activation of MEK/ERK pathways in both serum-free and serum-containing conditions, and this activation is required for the up-regulation of p21. Next, we performed immunofluorescence studies to assess whether the p21 accumulation induced by UCN-01 was nuclear or cytoplasmic and to test whether MEK blockade prevented not only the induction but also the redistribution of p21. As clearly demonstrated in Fig. 7 B, UCN-01 significantly increased p21 expression, similarly to what was observed previously at the Western blot level (Fig. 3). Moreover, the increase in p21 was exclusively nuclear as the immunostaining colo- calized with 4 Ј ,6-diamidino-2-phenylindole, a known nuclear fluores- cence stain, without evidence of redistribution to the cytoplasmic compartment (see Fig. 7 C, inset ). Furthermore, preincubation with the MEK inhibitor PD98059 significantly diminished the total expression of nuclear p21. Thus, induction of p21 by UCN-01 appears to be almost exclusively nuclear, and its expression requires the activation of MEK/ERK pathway. In summary, UCN-01 promotes cell cycle arrest through an increase in p21 protein expression. This increase is p53 independent and is due to transcriptional activation of the p21 waf1/cip1 promoter. The minimal promoter region of p21 is similar to the one activated by Ras, and p21 transcriptional activation by ...
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... (p21P-luc) by ϳ 5-fold. Similar activation ( ϳ 6-fold) was observed with Ras V12 (positive control). Moreover, we transfected HaCaT cell lines (a keratinocyte cell line with deficient p53 function; Ref. 31) with the p21P ⌬ p53-luc reporter, a p21 waf1/cip1 construct lacking 250 bases from the 5 Ј end corresponding to the p53 consensus DNA binding site. Again, UCN-01 activates this construct ( ϳ 6-fold) despite the lack of the p53 consensus site, indicating that neither the presence of p53 binding sites in the p21 waf1/cip1 promoter nor the presence of functional p53 protein was required for UCN-01 to activate the p21 waf1/cip1 promoter. To determine the minimal promoter region required for the transcriptional activation of p21 waf1/cip1 by UCN-01, additional promoter deletion constructs were tested. We initially used the p21PSma-luc construct (26, 30), which contains the p21 waf1/cip1 promoter sequences from base Ϫ 111 through the transcriptional initiation site. Of note, this construct represents the minimal promoter region activated by Ras (26, 30). As shown in Fig. 4, UCN-01 significantly activates this minimal promoter ( ϳ 7-fold), similar to Ras V12. Moreover, when HaCaT cells were transfected with full-length p21 waf1/cip1 construct lacking the Ras minimal promoter region p21PSma ⌬ 1-luc the induction was lost for both Ras V12 and UCN-01. Thus, the minimal p21 waf1/cip1 promoter region for UCN-01 is similar to that of Ras, and it seems to be represented by a region in the p21 waf1/cip1 promoter proximal to the Sma I site at Ϫ 111 (p21PSma-luc). To test whether the p21 waf1/cip1 transcriptional activation by UCN-01 occurs in other cell types, we transfected pSMA-luc into HCT116 isogenic cell lines (wild-type or p53 null cells). Again, UCN-01 activated pSMA-luc in both cell lines (data not shown), demonstrating that the induction of the p21 minimal promoter by UCN-01 occurs in other cell types and is also independent of p53 function. Require PKC nor PDK1/AKT Pathways. UCN-01 is a known inhibitor of several serine-threonine kinases, including PKC and PDK1 (14, 32, 33). To test whether the p21 transcriptional effects of UCN-01 might be related to PKC modulation, we assessed whether the putative effects of UCN-01 on PKC function occur at times and concentrations relevant for the p21 induction induced by this agent. To this end, we assessed the phosphorylation of adducin, a known substrate of PKC (16, 34). As demonstrated in Fig. 5 A, the phosphorylation of adducin was unaltered even at the highest UCN-01 concentrations tested. Thus, the putative effects of UCN-01 in PKC activity appear not to be relevant for p21 transcription, as demonstrated previously with respect to apoptosis and cell cycle arrest (15, 17). Sato et al. (33) demonstrated recently that UCN-01 inhibits PDK1, a serine-threonine kinase responsible for the activation of AKT (35, 36). Of note, fully active AKT requires the phosphorylation of 2 residues, threonine 308 (site phosphorylated by PDK1) and serine 473 (site phosphorylated by an undefined kinase; Refs. 35, 36). To assess whether UCN-01 activates p21 transcription by modulation of AKT signaling, we tested whether UCN-01 promotes the loss in AKT phosphorylation in HaCaT cell lines. As shown in Fig. 5 A, although UCN-01 induced p21 at concentrations Ͼ 30 n M (as shown before), the effects of UCN-01 on dephosphorylation of AKT on site S473 (PDK- independent site) occurred only at concentrations Ͼ 300 n M . Moreover, the PDK1-dependent site, threonine 308, was unchanged at the times and concentrations required for p21 induction by UCN-01. In contrast, the small molecule PI3k inhibitor LY294002 dephosphoryl- ated AKT at both residues (threonine 308 and serine 473) while having minimal effects on p21 expression (Fig. 5 A ). Similar results were observed with another chemically unrelated PI3k inhibitor, wortmannin (data not shown). Furthermore, coexposure of HaCaT cells to UCN-01 in combination with either LY294002 or wortmannin showed unaltered induction of p21 (data not shown). These results suggest that induction of p21 by UCN-01 is not related to the capacity of UCN-01 to inhibit PI3k/AKT pathway. To additionally exclude the possibility that the effects of UCN-01 in the PDK1/AKT pathway might be responsible for p21 up-regulation, we expressed a constitutively active form of Akt (Myr-Akt) and a kinase-inactive form of Akt (T308A and S473A) in HaCaT cells using adenoviral delivery. Upon infection with increasing multiplicity of infection of the AKT AdVs, a dose-dependent increase in the expression of the corresponding proteins was detected, as verified by Western blot using an antibody against total AKT (Fig. 5 B ). Then, HaCaT cells infected with myr-AKT adenovirus were exposed to UCN-01. Consistent with Fig. 3, p21 was significantly increased in HaCaT cells exposed to UCN-01 (Fig. 5 B, Lane 6 ). Increasing multiplicity of infection of activated AKT failed to prevent this up- regulation (Fig. 5 B, Lanes 7–10 ). Moreover, increasing multiplicity of infection with kinase-deficient AKT failed to increase p21, strongly suggesting that the effects of UCN-01 in p21 expression appear not to be related to the effects on the PDK1/AKT pathway reported previously. Activation of the MEK/ERK Pathway. Because the transcriptional up-regulation of p21 by UCN-01 is p53 independent and the minimal promoter region is similar to the one required for the proto-oncogene Ras to activate the p21 promoter, we tested whether UCN-01 could modulate signal pathways downstream to Ras. Ras modulates transcriptional events by activating the mitogen-activated protein kinase (MAPK) kinase kinase, Raf (37 – 40). In turn, Raf activates MEK and this, in turn, phosphorylates and activates the ERK1 and ERK2, leading to the phosphorylation and activation of several transcriptional factors. To test whether UCN-01 activates the MEK/ERK pathway, we exposed serum-starved HaCaT cells to increasing concentrations of UCN-01 for 60 min or for increasing time periods followed by in vitro kinase reactions. UCN-01 at concentrations Ն 100 n M significantly activated ERK kinase activity (Fig. 6 A ). Similar results were obtained when protein lysates were immunoblotted with a phospho-specific ERK1/2 antibody (data not shown). Time course analysis revealed that 30 min of 100 n M UCN-01 exposure were sufficient to activate the MEK/ERK pathway (Fig. 6 A, right ). Thus, MAPK activation occurs at similar concentrations ( Ն 100 n M ) required for p21 up-regulation. Moreover, as demonstrated in Fig. 6 A, the activation of MAPK temporally precedes the increase in p21 mRNA (3 – 6 h; Fig. 3 C ). To confirm the involvement of the MEK/ERK pathway in UCN-01-induced p21 transcriptional up-regulation, we used two structurally unrelated chemical inhibitors of the MAPK kinase, MEK (PD98059 and U0126), at concentrations that were specific for MEK inhibition (41, 42). HaCaT cells either exposed to UCN-01 (Fig. 6 B, Lane 2 ) or transfected with Ras (Fig. 6 B, Lane 5 ) demonstrated an increase in p21 promoter activity, as previously shown in Fig. 4. However, cotransfection with dominant-negative MEK (MEKAA) or preincubation with the MEK inhibitor PD98059 blunted the activation of the p21 promoter by either UCN-01 or Ras. Similar results were obtained when p21 expression was determined by Northern blot studies (Fig. 6 C ). Activation of the MEK/ERK Pathway. To test whether MAPK was also required for the induction of p21 at the protein level, serum- starved HaCaT cells treated with UCN-01 were preincubated with the chemical MEK inhibitor PD98059. Again, the loss in MEK activity induced by PD98059, as measured by loss in ERK phosphorylation, blunted the p21 protein induction induced by UCN-01 (Fig. 7 A ). Moreover, we assessed whether the activation of MEK occurs also in serum containing exponentially growing conditions. To this end, subconfluent HaCaT cells treated with UCN-01 were preincubated with another structurally unrelated MEK inhibitor, UO126. As clearly demonstrated in Fig. 7 B, the induction of p21 by UCN-01 was associated with activation of MEK, as measured by antiserum that only recognizes phosphorylated MEK. Moreover, preincubation with the MEK inhibitor blunted the induction of p21 by UCN-01. Together, UCN-01 induces the activation of MEK/ERK pathways in both serum-free and serum-containing conditions, and this activation is required for the up-regulation of p21. Next, we performed immunofluorescence studies to assess whether the p21 accumulation induced by UCN-01 was nuclear or cytoplasmic and to test whether MEK blockade prevented not only the induction but also the redistribution of p21. As clearly demonstrated in Fig. 7 B, UCN-01 significantly increased p21 expression, similarly to what was observed previously at the Western blot level (Fig. 3). Moreover, the increase in p21 was exclusively nuclear as the immunostaining colo- calized with 4 Ј ,6-diamidino-2-phenylindole, a known nuclear fluores- cence stain, without evidence of redistribution to the cytoplasmic compartment (see Fig. 7 C, inset ). Furthermore, preincubation with the MEK inhibitor PD98059 significantly diminished the total expression of nuclear p21. Thus, induction of p21 by UCN-01 appears to be almost exclusively nuclear, and its expression requires the activation of MEK/ERK pathway. In summary, UCN-01 promotes cell cycle arrest through an increase in p21 protein expression. This increase is p53 independent and is due to transcriptional activation of the p21 waf1/cip1 promoter. The minimal promoter region of p21 is similar to the one activated by Ras, and p21 transcriptional activation by UCN-01 requires the activation of the MEK/MAPK pathway. This finding contributes to the under- standing of the mechanism of cell cycle arrest by this compound and may help also in designing more adequate clinical trials with this agent. In this report, we demonstrate, for the first time, that ...
Context 5
... that concentrations Ͼ 30 n M UCN-01 for 12 h promoted the accumulation of p21 protein. This accumulation occurs as early as 3 h after exposure to 100 n M UCN-01 concentrations. Again, in parallel samples, the expression of p27 kip1 did not increase. This result confirms our previous observation using the isogenic HCT116 model (Fig. 2), suggesting that accumulation of p27 kip1 is not required for UCN-01-induced cell cycle arrest. crease in p21 mRNA Steady-State Levels. On the basis of the biological relevance of p21 in UCN-01-induced cell cycle arrest, we focused on the mechanism by which UCN-01 induces this endogenous CDK inhibitor. To this end, we asked whether p21 protein accumulation by UCN-01 is the result of an increase in p21 mRNA. Dose-response analyses (Fig. 3 C ) and time course studies (Fig. 3 D ) demonstrated that p21 mRNA increases by 3 h, at concentrations Ն 100 n M , as measured by Northern blot analysis, similar to the concentrations necessary for the increase in p21 protein levels as measured by Western blot analysis (see Fig. 3 A ). Thus, the increase in p21 mRNA by UCN-01 occurs at similar times and concentrations required for the increase in p21 protein expression. To determine whether UCN-01-induced accumulation of p21 waf1/cip1 was due to post-transcriptional control, we conducted pulse-chase analysis in the presence of actinomycin D and measured p21 mRNA by Northern blot studies. We observed that p21 mRNA expression decreased to a similar extent in both vehicle and UCN-01-treated cells up to 6 h after actinomycin D (85% and 82%, respectively). These experiments demonstrate that p21 accumulation promoted by UCN-01 is not explained by increased in p21 mRNA half-life. Instead, it appears to be due to transcriptional effects. quired for Ras to Activate the p21 Promoter. To confirm that the increase in p21 waf1/cip1 mRNA induced by UCN-01 was due to transcriptional activation of the p21 waf1/cip1 promoter, we transiently transfected HaCaT cells with several p21 waf1/cip1 promoter-driven luciferase reporter plasmids (26, 30). Twelve h after transfection, HaCaT cells were exposed to 100 n M UCN-01 for 18 h, and luciferase activity was measured as described in “ Materials and Methods. ” As a positive control, we transiently transfected HaCaT cells with activated Ras (Ras V12), a known transcriptional activator of the p21 waf1/cip1 promoter (26). As shown in Fig. 4, UCN-01 significantly enhanced the transcriptional activity of the p21 full-length 2.4 kb promoter (p21P-luc) by ϳ 5-fold. Similar activation ( ϳ 6-fold) was observed with Ras V12 (positive control). Moreover, we transfected HaCaT cell lines (a keratinocyte cell line with deficient p53 function; Ref. 31) with the p21P ⌬ p53-luc reporter, a p21 waf1/cip1 construct lacking 250 bases from the 5 Ј end corresponding to the p53 consensus DNA binding site. Again, UCN-01 activates this construct ( ϳ 6-fold) despite the lack of the p53 consensus site, indicating that neither the presence of p53 binding sites in the p21 waf1/cip1 promoter nor the presence of functional p53 protein was required for UCN-01 to activate the p21 waf1/cip1 promoter. To determine the minimal promoter region required for the transcriptional activation of p21 waf1/cip1 by UCN-01, additional promoter deletion constructs were tested. We initially used the p21PSma-luc construct (26, 30), which contains the p21 waf1/cip1 promoter sequences from base Ϫ 111 through the transcriptional initiation site. Of note, this construct represents the minimal promoter region activated by Ras (26, 30). As shown in Fig. 4, UCN-01 significantly activates this minimal promoter ( ϳ 7-fold), similar to Ras V12. Moreover, when HaCaT cells were transfected with full-length p21 waf1/cip1 construct lacking the Ras minimal promoter region p21PSma ⌬ 1-luc the induction was lost for both Ras V12 and UCN-01. Thus, the minimal p21 waf1/cip1 promoter region for UCN-01 is similar to that of Ras, and it seems to be represented by a region in the p21 waf1/cip1 promoter proximal to the Sma I site at Ϫ 111 (p21PSma-luc). To test whether the p21 waf1/cip1 transcriptional activation by UCN-01 occurs in other cell types, we transfected pSMA-luc into HCT116 isogenic cell lines (wild-type or p53 null cells). Again, UCN-01 activated pSMA-luc in both cell lines (data not shown), demonstrating that the induction of the p21 minimal promoter by UCN-01 occurs in other cell types and is also independent of p53 function. Require PKC nor PDK1/AKT Pathways. UCN-01 is a known inhibitor of several serine-threonine kinases, including PKC and PDK1 (14, 32, 33). To test whether the p21 transcriptional effects of UCN-01 might be related to PKC modulation, we assessed whether the putative effects of UCN-01 on PKC function occur at times and concentrations relevant for the p21 induction induced by this agent. To this end, we assessed the phosphorylation of adducin, a known substrate of PKC (16, 34). As demonstrated in Fig. 5 A, the phosphorylation of adducin was unaltered even at the highest UCN-01 concentrations tested. Thus, the putative effects of UCN-01 in PKC activity appear not to be relevant for p21 transcription, as demonstrated previously with respect to apoptosis and cell cycle arrest (15, 17). Sato et al. (33) demonstrated recently that UCN-01 inhibits PDK1, a serine-threonine kinase responsible for the activation of AKT (35, 36). Of note, fully active AKT requires the phosphorylation of 2 residues, threonine 308 (site phosphorylated by PDK1) and serine 473 (site phosphorylated by an undefined kinase; Refs. 35, 36). To assess whether UCN-01 activates p21 transcription by modulation of AKT signaling, we tested whether UCN-01 promotes the loss in AKT phosphorylation in HaCaT cell lines. As shown in Fig. 5 A, although UCN-01 induced p21 at concentrations Ͼ 30 n M (as shown before), the effects of UCN-01 on dephosphorylation of AKT on site S473 (PDK- independent site) occurred only at concentrations Ͼ 300 n M . Moreover, the PDK1-dependent site, threonine 308, was unchanged at the times and concentrations required for p21 induction by UCN-01. In contrast, the small molecule PI3k inhibitor LY294002 dephosphoryl- ated AKT at both residues (threonine 308 and serine 473) while having minimal effects on p21 expression (Fig. 5 A ). Similar results were observed with another chemically unrelated PI3k inhibitor, wortmannin (data not shown). Furthermore, coexposure of HaCaT cells to UCN-01 in combination with either LY294002 or wortmannin showed unaltered induction of p21 (data not shown). These results suggest that induction of p21 by UCN-01 is not related to the capacity of UCN-01 to inhibit PI3k/AKT pathway. To additionally exclude the possibility that the effects of UCN-01 in the PDK1/AKT pathway might be responsible for p21 up-regulation, we expressed a constitutively active form of Akt (Myr-Akt) and a kinase-inactive form of Akt (T308A and S473A) in HaCaT cells using adenoviral delivery. Upon infection with increasing multiplicity of infection of the AKT AdVs, a dose-dependent increase in the expression of the corresponding proteins was detected, as verified by Western blot using an antibody against total AKT (Fig. 5 B ). Then, HaCaT cells infected with myr-AKT adenovirus were exposed to UCN-01. Consistent with Fig. 3, p21 was significantly increased in HaCaT cells exposed to UCN-01 (Fig. 5 B, Lane 6 ). Increasing multiplicity of infection of activated AKT failed to prevent this up- regulation (Fig. 5 B, Lanes 7–10 ). Moreover, increasing multiplicity of infection with kinase-deficient AKT failed to increase p21, strongly suggesting that the effects of UCN-01 in p21 expression appear not to be related to the effects on the PDK1/AKT pathway reported previously. Activation of the MEK/ERK Pathway. Because the transcriptional up-regulation of p21 by UCN-01 is p53 independent and the minimal promoter region is similar to the one required for the proto-oncogene Ras to activate the p21 promoter, we tested whether UCN-01 could modulate signal pathways downstream to Ras. Ras modulates transcriptional events by activating the mitogen-activated protein kinase (MAPK) kinase kinase, Raf (37 – 40). In turn, Raf activates MEK and this, in turn, phosphorylates and activates the ERK1 and ERK2, leading to the phosphorylation and activation of several transcriptional factors. To test whether UCN-01 activates the MEK/ERK pathway, we exposed serum-starved HaCaT cells to increasing concentrations of UCN-01 for 60 min or for increasing time periods followed by in vitro kinase reactions. UCN-01 at concentrations Ն 100 n M significantly activated ERK kinase activity (Fig. 6 A ). Similar results were obtained when protein lysates were immunoblotted with a phospho-specific ERK1/2 antibody (data not shown). Time course analysis revealed that 30 min of 100 n M UCN-01 exposure were sufficient to activate the MEK/ERK pathway (Fig. 6 A, right ). Thus, MAPK activation occurs at similar concentrations ( Ն 100 n M ) required for p21 up-regulation. Moreover, as demonstrated in Fig. 6 A, the activation of MAPK temporally precedes the increase in p21 mRNA (3 – 6 h; Fig. 3 C ). To confirm the involvement of the MEK/ERK pathway in UCN-01-induced p21 transcriptional up-regulation, we used two structurally unrelated chemical inhibitors of the MAPK kinase, MEK (PD98059 and U0126), at concentrations that were specific for MEK inhibition (41, 42). HaCaT cells either exposed to UCN-01 (Fig. 6 B, Lane 2 ) or transfected with Ras (Fig. 6 B, Lane 5 ) demonstrated an increase in p21 promoter activity, as previously shown in Fig. 4. However, cotransfection with dominant-negative MEK (MEKAA) or preincubation with the MEK inhibitor PD98059 blunted the activation of the p21 promoter by either UCN-01 or Ras. Similar results were obtained when p21 expression was determined by Northern blot studies (Fig. 6 C ). Activation of the MEK/ERK Pathway. To test whether MAPK ...
Context 6
... studies (Fig. 3 D ) demonstrated that p21 mRNA increases by 3 h, at concentrations Ն 100 n M , as measured by Northern blot analysis, similar to the concentrations necessary for the increase in p21 protein levels as measured by Western blot analysis (see Fig. 3 A ). Thus, the increase in p21 mRNA by UCN-01 occurs at similar times and concentrations required for the increase in p21 protein expression. To determine whether UCN-01-induced accumulation of p21 waf1/cip1 was due to post-transcriptional control, we conducted pulse-chase analysis in the presence of actinomycin D and measured p21 mRNA by Northern blot studies. We observed that p21 mRNA expression decreased to a similar extent in both vehicle and UCN-01-treated cells up to 6 h after actinomycin D (85% and 82%, respectively). These experiments demonstrate that p21 accumulation promoted by UCN-01 is not explained by increased in p21 mRNA half-life. Instead, it appears to be due to transcriptional effects. quired for Ras to Activate the p21 Promoter. To confirm that the increase in p21 waf1/cip1 mRNA induced by UCN-01 was due to transcriptional activation of the p21 waf1/cip1 promoter, we transiently transfected HaCaT cells with several p21 waf1/cip1 promoter-driven luciferase reporter plasmids (26, 30). Twelve h after transfection, HaCaT cells were exposed to 100 n M UCN-01 for 18 h, and luciferase activity was measured as described in “ Materials and Methods. ” As a positive control, we transiently transfected HaCaT cells with activated Ras (Ras V12), a known transcriptional activator of the p21 waf1/cip1 promoter (26). As shown in Fig. 4, UCN-01 significantly enhanced the transcriptional activity of the p21 full-length 2.4 kb promoter (p21P-luc) by ϳ 5-fold. Similar activation ( ϳ 6-fold) was observed with Ras V12 (positive control). Moreover, we transfected HaCaT cell lines (a keratinocyte cell line with deficient p53 function; Ref. 31) with the p21P ⌬ p53-luc reporter, a p21 waf1/cip1 construct lacking 250 bases from the 5 Ј end corresponding to the p53 consensus DNA binding site. Again, UCN-01 activates this construct ( ϳ 6-fold) despite the lack of the p53 consensus site, indicating that neither the presence of p53 binding sites in the p21 waf1/cip1 promoter nor the presence of functional p53 protein was required for UCN-01 to activate the p21 waf1/cip1 promoter. To determine the minimal promoter region required for the transcriptional activation of p21 waf1/cip1 by UCN-01, additional promoter deletion constructs were tested. We initially used the p21PSma-luc construct (26, 30), which contains the p21 waf1/cip1 promoter sequences from base Ϫ 111 through the transcriptional initiation site. Of note, this construct represents the minimal promoter region activated by Ras (26, 30). As shown in Fig. 4, UCN-01 significantly activates this minimal promoter ( ϳ 7-fold), similar to Ras V12. Moreover, when HaCaT cells were transfected with full-length p21 waf1/cip1 construct lacking the Ras minimal promoter region p21PSma ⌬ 1-luc the induction was lost for both Ras V12 and UCN-01. Thus, the minimal p21 waf1/cip1 promoter region for UCN-01 is similar to that of Ras, and it seems to be represented by a region in the p21 waf1/cip1 promoter proximal to the Sma I site at Ϫ 111 (p21PSma-luc). To test whether the p21 waf1/cip1 transcriptional activation by UCN-01 occurs in other cell types, we transfected pSMA-luc into HCT116 isogenic cell lines (wild-type or p53 null cells). Again, UCN-01 activated pSMA-luc in both cell lines (data not shown), demonstrating that the induction of the p21 minimal promoter by UCN-01 occurs in other cell types and is also independent of p53 function. Require PKC nor PDK1/AKT Pathways. UCN-01 is a known inhibitor of several serine-threonine kinases, including PKC and PDK1 (14, 32, 33). To test whether the p21 transcriptional effects of UCN-01 might be related to PKC modulation, we assessed whether the putative effects of UCN-01 on PKC function occur at times and concentrations relevant for the p21 induction induced by this agent. To this end, we assessed the phosphorylation of adducin, a known substrate of PKC (16, 34). As demonstrated in Fig. 5 A, the phosphorylation of adducin was unaltered even at the highest UCN-01 concentrations tested. Thus, the putative effects of UCN-01 in PKC activity appear not to be relevant for p21 transcription, as demonstrated previously with respect to apoptosis and cell cycle arrest (15, 17). Sato et al. (33) demonstrated recently that UCN-01 inhibits PDK1, a serine-threonine kinase responsible for the activation of AKT (35, 36). Of note, fully active AKT requires the phosphorylation of 2 residues, threonine 308 (site phosphorylated by PDK1) and serine 473 (site phosphorylated by an undefined kinase; Refs. 35, 36). To assess whether UCN-01 activates p21 transcription by modulation of AKT signaling, we tested whether UCN-01 promotes the loss in AKT phosphorylation in HaCaT cell lines. As shown in Fig. 5 A, although UCN-01 induced p21 at concentrations Ͼ 30 n M (as shown before), the effects of UCN-01 on dephosphorylation of AKT on site S473 (PDK- independent site) occurred only at concentrations Ͼ 300 n M . Moreover, the PDK1-dependent site, threonine 308, was unchanged at the times and concentrations required for p21 induction by UCN-01. In contrast, the small molecule PI3k inhibitor LY294002 dephosphoryl- ated AKT at both residues (threonine 308 and serine 473) while having minimal effects on p21 expression (Fig. 5 A ). Similar results were observed with another chemically unrelated PI3k inhibitor, wortmannin (data not shown). Furthermore, coexposure of HaCaT cells to UCN-01 in combination with either LY294002 or wortmannin showed unaltered induction of p21 (data not shown). These results suggest that induction of p21 by UCN-01 is not related to the capacity of UCN-01 to inhibit PI3k/AKT pathway. To additionally exclude the possibility that the effects of UCN-01 in the PDK1/AKT pathway might be responsible for p21 up-regulation, we expressed a constitutively active form of Akt (Myr-Akt) and a kinase-inactive form of Akt (T308A and S473A) in HaCaT cells using adenoviral delivery. Upon infection with increasing multiplicity of infection of the AKT AdVs, a dose-dependent increase in the expression of the corresponding proteins was detected, as verified by Western blot using an antibody against total AKT (Fig. 5 B ). Then, HaCaT cells infected with myr-AKT adenovirus were exposed to UCN-01. Consistent with Fig. 3, p21 was significantly increased in HaCaT cells exposed to UCN-01 (Fig. 5 B, Lane 6 ). Increasing multiplicity of infection of activated AKT failed to prevent this up- regulation (Fig. 5 B, Lanes 7–10 ). Moreover, increasing multiplicity of infection with kinase-deficient AKT failed to increase p21, strongly suggesting that the effects of UCN-01 in p21 expression appear not to be related to the effects on the PDK1/AKT pathway reported previously. Activation of the MEK/ERK Pathway. Because the transcriptional up-regulation of p21 by UCN-01 is p53 independent and the minimal promoter region is similar to the one required for the proto-oncogene Ras to activate the p21 promoter, we tested whether UCN-01 could modulate signal pathways downstream to Ras. Ras modulates transcriptional events by activating the mitogen-activated protein kinase (MAPK) kinase kinase, Raf (37 – 40). In turn, Raf activates MEK and this, in turn, phosphorylates and activates the ERK1 and ERK2, leading to the phosphorylation and activation of several transcriptional factors. To test whether UCN-01 activates the MEK/ERK pathway, we exposed serum-starved HaCaT cells to increasing concentrations of UCN-01 for 60 min or for increasing time periods followed by in vitro kinase reactions. UCN-01 at concentrations Ն 100 n M significantly activated ERK kinase activity (Fig. 6 A ). Similar results were obtained when protein lysates were immunoblotted with a phospho-specific ERK1/2 antibody (data not shown). Time course analysis revealed that 30 min of 100 n M UCN-01 exposure were sufficient to activate the MEK/ERK pathway (Fig. 6 A, right ). Thus, MAPK activation occurs at similar concentrations ( Ն 100 n M ) required for p21 up-regulation. Moreover, as demonstrated in Fig. 6 A, the activation of MAPK temporally precedes the increase in p21 mRNA (3 – 6 h; Fig. 3 C ). To confirm the involvement of the MEK/ERK pathway in UCN-01-induced p21 transcriptional up-regulation, we used two structurally unrelated chemical inhibitors of the MAPK kinase, MEK (PD98059 and U0126), at concentrations that were specific for MEK inhibition (41, 42). HaCaT cells either exposed to UCN-01 (Fig. 6 B, Lane 2 ) or transfected with Ras (Fig. 6 B, Lane 5 ) demonstrated an increase in p21 promoter activity, as previously shown in Fig. 4. However, cotransfection with dominant-negative MEK (MEKAA) or preincubation with the MEK inhibitor PD98059 blunted the activation of the p21 promoter by either UCN-01 or Ras. Similar results were obtained when p21 expression was determined by Northern blot studies (Fig. 6 C ). Activation of the MEK/ERK Pathway. To test whether MAPK was also required for the induction of p21 at the protein level, serum- starved HaCaT cells treated with UCN-01 were preincubated with the chemical MEK inhibitor PD98059. Again, the loss in MEK activity induced by PD98059, as measured by loss in ERK phosphorylation, blunted the p21 protein induction induced by UCN-01 (Fig. 7 A ). Moreover, we assessed whether the activation of MEK occurs also in serum containing exponentially growing conditions. To this end, subconfluent HaCaT cells treated with UCN-01 were preincubated with another structurally unrelated MEK inhibitor, UO126. As clearly demonstrated in Fig. 7 B, the induction of p21 by UCN-01 was associated with activation of MEK, as measured by antiserum that only recognizes phosphorylated MEK. Moreover, preincubation with the MEK inhibitor ...
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... As shown in Fig. 1d, AZD7762 modified only Chk1 expression level which is consistent with other studies 36 while UCN-01 additionally downregulated total RB and pRB levels at concentrations >20 nM without affecting E2F expression (Fig. 1d). UCN-01, in contrast to AZD7762, induces G1-S phase arrest in the cell cycle 36,37 . This off-target phenomenon of UCN-01 has been documented before as it also targets several other CDKs, AKT, and protein kinase C 38,39 . ...
CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.
... Third, analogous to other pure antiestrogens, C75 treatment resulted in ERα protein downregulation while apparently promoting MAPK activation and ERα transactivation. Considering previous studies showing that activation of MAPK/ERK signaling can induce p21 WAF1/CIP1 expression and that ERα is a known transcriptional regulator of p21 WAF1/CIP1 , [67][68][69] additional studies are warranted to investigate how MAPK/ERK and ERα might synergistically regulate C75-induced p21 WAF1/CIP1 expression. Although the precise mechanism of C75-mediated reduction of ERα protein remains unclear, it is plausible that loss of FASN signaling induces proteasome-dependent ERα degradation and/or epigenetic inhibition of ERα mRNA synthesis, driving target cells to completely different outcomes to those induced when E 2 binding acts as the natural signal for ERα degradation and the parallel reduction in ERα mRNA levels. ...
Fatty acid synthase (FASN), the key enzyme for endogenous synthesis of fatty acids, is overexpressed and hyperactivated in a biologically aggressive subset of sex steroid-related tumors, including breast carcinomas. Using pharmacological and genetic approaches, we assessed the molecular relationship between FASN signaling and estrogen receptor alpha (ERα) signaling in breast cancer. The small compound C75, a synthetic slow-binding inhibitor of FASN activity, induced a dramatic augmentation of estradiol (E2)-stimulated, ERα-driven transcription. FASN and ERα were both necessary for the synergistic activation of ERα transcriptional activity that occurred following co-exposure to C75 and E2: first, knockdown of FASN expression using RNAi (RNA interference) drastically lowered (>100 fold) the amount of E2 required for optimal activation of ERα-mediated transcriptional activity; second, FASN blockade synergistically increased E2-stimulated ERα-mediated transcriptional activity in ERα-negative breast cancer cells stably transfected with ERα, but not in ERα-negative parental cells. Non-genomic, E2-regulated cross-talk between the ERα and MAPK pathways participated in these phenomena. Thus, treatment with the pure antiestrogen ICI 182 780 or the potent and specific inhibitor of MEK/ERK, U0126, was sufficient to abolish the synergistic nature of the interaction between FASN blockade and E2-stimulated ERα transactivation. FASN inhibition suppressed E2-stimulated breast cancer cell proliferation and anchorage-independent colony formation while promoting the reduction of ERα protein. FASN blockade resulted in the increased expression and nuclear accumulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, two critical mediators of the therapeutic effects of antiestrogen in breast cancer, while inactivating AKT, a key mediator of E2-promoted anchorage-independent growth. The ability of FASN to regulate E2/ERα signaling may represent a promising strategy for anticancer treatment involving a new generation of FASN inhibitors.
... The UCN-01 (7-Hidroxystaurosporine) is a staurosporine analog isolated from the culture broth of Streptomyces species, as indicated in [81][82][83][84]. The UCN-01 induced the cell cycle arrest in G1/S phase, induction of CDKN1A (p21WAF1) and dephosphorylation of both CDK2 and RB proteins [81][82][83][84]. ...
... The UCN-01 (7-Hidroxystaurosporine) is a staurosporine analog isolated from the culture broth of Streptomyces species, as indicated in [81][82][83][84]. The UCN-01 induced the cell cycle arrest in G1/S phase, induction of CDKN1A (p21WAF1) and dephosphorylation of both CDK2 and RB proteins [81][82][83][84]. The UCN-01 can also inhibit the activity of CDK1 and CDK2 [81]. ...
... This inhibition leads to activation of cyclin B/CDK1 and marked abrogation of the G2-phase point of control, promotion of the cells to undergo mitosis and early apoptotic death later, instead of cell cycle arrest [81,82]. Several phase I studies have been conducted with UCN-01, both as a single agent and in combination with cytotoxic chemotherapy [81][82][83]. ...
Natural compounds from various plants, microorganisms and marine species play an important role in the discovery novel components that can be successfully used in numerous biomedical applications, including anticancer therapeutics. Since uncontrolled and rapid cell division is a hallmark of cancer, unraveling the molecular mechanisms underlying mitosis is key to understanding how various natural compounds might function as inhibitors of cell cycle progression. A number of natural compounds that inhibit the cell cycle arrest have proven effective for killing cancer cells in vitro, in vivo and in clinical settings. Significant advances that have been recently made in the understanding of molecular mechanisms underlying the cell cycle regulation using the chemotherapeutic agents is of great importance for improving the efficacy of targeted therapeutics and overcoming resistance to anticancer drugs, especially of natural origin, which inhibit the activities of cyclins and cyclin-dependent kinases, as well as other proteins and enzymes involved in proper regulation of cell cycle leading to controlled cell proliferation.
... As shown in Fig. 1d, AZD7762 modified only Chk1 expression level which is consistent with other studies 36 while UCN-01 additionally downregulated total RB and pRB levels at concentrations >20 nM without affecting E2F expression (Fig. 1d). UCN-01, in contrast to AZD7762, induces G1-S phase arrest in the cell cycle 36,37 . This off-target phenomenon of UCN-01 has been documented before as it also targets several other CDKs, AKT, and protein kinase C 38,39 . ...
Therapeutic intervention using oncolytic viruses is called virotherapy. This type of virus is defined by the ability to replicate in tumor cells only and to destroy these cells upon replication. In addition, this virus type is able to induce a tumor-directed immune response. Early clinical trials have confirmed the safety profile of oncolytic viruses. Currently, different groups are working on the development of oncolytic viruses with a focus on treatment of nonmuscle invasive bladder cancer (NMIBC). A preliminary active recruiting clinical phase II/III trial ongoing in patients with a NMIBC was recently implemented in the United States. Our research group developed an oncolytic adenovirus that will soon enter a clinical phase I trial in patients diagnosed with glioma. This virus is being further modified for the treatment of NMIBC. In this review article, recent developments in the design and use of virotherapy in bladder cancer are summarized.
... For example, ellipticine, P53 Reactivation and Induction of Massive Apoptosis (PRIMA-1), and Mutant p53 reactivation and Induction of Rapid Apoptosis (MIRA-1) have been shown to induce a conformational change of mutant p53 that reverts the protein to wild type activity. Interestingly, UCN01, a bifunctional modulator of cyclin-dependent kinases, can arrest cancer cells harboring mutant p53 at the G2 checkpoint, leading to cell death (Facchinetti et al., 2004). ...
Apoptosis and autophagy are physiologically necessary pathways that are vital for cell homeostasis. Apoptosis facilitates type I programmed cell death, while the autophagic survival mechanism counteracts apoptosis. Dysregulation in the homeostatic balance between these two essential cellular pathways has been linked to various diseases. We review relevant Janus molecules and their interactomes, as well as lysosomes which play multiple roles in apoptosis and autophagy, and discuss how targeted interventions can be used in cancer prevention and/or therapy.
... UCN-01 (7-hydroxystaurosporine) inhibits several protein kinases including PKC and cyclin-dependent kinases, resulting in dysregulation of cell cycle and induction of apoptosis [35,36]. Clinical trials with UCN-01 as a single anticancer agent or in combination with other anticancer agents have been completed or are currently active for a wide range of cancers including T-cell lymphomas, leukemia, advanced kidney cancers, melanoma, and advanced solid tumors such as pancreatic, colorectal, and nonsmall cells lung cancers [37][38][39][40][41]. ...
Oncogenic mutant K-Ras is highly prevalent in multiple human tumors. Despite significant efforts to directly target Ras activity, no K-Ras-specific inhibitors have been developed and taken into the clinic. Since Ras proteins must be anchored to the inner leaflet of the plasma membrane (PM) for full biological activity, we devised a high-content screen to identify molecules with ability to displace K-Ras from the PM. Here we summarize the biochemistry and biology of three classes of compound identified by this screening method that inhibit K-Ras PM targeting: staurosporine and analogs, fendiline, and metformin. All three classes of compound significantly abrogate cell proliferation and Ras signaling in K-Ras-transformed cancer cells. Taken together, these studies provide an important proof of concept that blocking PM localization of K-Ras is a tractable therapeutic target.
... The phosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) is critical in breast cancer development and therapy due to a frequent mutation in this pathway that causes hyperactivation and promotes cell cycle progression (26)(27)(28). Although the mitogen-activated protein kinaseextracellular signal-regulated kinase (MAPK-ERK) pathway is generally associated with cell proliferation (29), increasing evidence indicates that prolonged hyperactivation of ERK leads to p21 Cip1/Waf1 upregulation, cell cycle arrest and cell senescence (23,30). ...
... Activation of MAPK signaling can induce p21 Cip1/Waf1 expression and cell cycle arrest/cell senescence (30). To determine if the MEK/ERK pathway is involved in BNF-induced p21 Cip1/Waf1 transcriptional up-regulation, we exposed MCF-7 cells to the MEK inhibitor PD98059 (10 µM). ...
... Activation of the MAPK pathway is generally associated with increased cell proliferation (29), but increasing studies have shown that the prolonged MAPK/ERK activation can induce p21 Cip1/Waf1 expression, leading to cell cycle arrest and cell senescence (23,30). AhR-MAPK crosstalk has been reported; those studies indicate that TCDD activates ERK via an AhRdependent or independent pathway, but TCDD-stimulated ERK appears only to affect AhR activity, with no effects on the ERK-mediated downstream transcriptional activity (41). ...
Beta-naphthoflavone (BNF, DB06732) is an agonist of aryl hydrocarbon receptor (AhR) and a putative chemotherapeutic agent that has antitumor activity against mammary carcinomas in vivo. However, the mechanism by which BNF exerts this antitumor effect remains unclear. Thus, we explored mechanisms of BNF's antitumor effects in human breast cancer cells. BNF suppressed cell proliferation and induced cell cycle arrest in the G0/G1 phase with downregulation of cyclin D1/D3 and CDK4 and upregulation of p21(Cip1/Waf1), leading to a senescence-like phenotype in estrogen receptor (ER)-positive MCF-7 cells, but not in ER-negative MDA-MB-231 cells. In addition, BNF inhibited PI3K/AKT signaling, and the PI3K inhibitor, LY294,002, exhibited the same inhibitory effects on cyclinD1/D3, CDK4 and the cell cycle as BNF. Interestingly, BNF activated mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK-ERK) signaling, and more notably, the MEK inhibitor PD98059 significantly blocked BNF-induced cell cycle arrest and upregulation of p21(Cip1/Waf1). Furthermore, specific ERα and AhR siRNA studies indicate that ERα is required in BNF-induced p21(Cip1/Waf1) expression, and BNF-mediated cell cycle arrest and modulation of AKT and ERK signaling is AhR dependent. Taken together, AhR-dependent inhibition of the PI3K/AKT pathway, activation of MAPK/ERK and modulation of ERα is a novel mechanism underlying BNF-mediated antitumor effects in breast cancer, which may represent a promising strategy to be exploited in future clinical trials.
... Although p21 is a prominent transcriptional target of p53 another pathway must have triggered the p21 increase since HL-60 cells are p53-deficient (25). As also the activation of the MEK-Erk pathway was shown to up-regulate p21 (26,27), we checked the phosphorylation status of Erk1/2. S. floribunda showed a slight increase of the phosphorylation status of Erk1/2 at the 4-h time-point going along with the p21 up-regulation. ...
Different studies describe the anti-inflammatory effects of Scrophularia species, a medicinal plant widely used in folk medicine since ancient times. As knowledge regarding the anti-neoplastic properties of this species is rather limited, we investigated the influence of methanol extracts of different Scrophularia species on cell proliferation, cell death, and tumour cell intravasation through the lymph endothelial barrier. HL-60 leukaemia cells were treated with methanol extracts of different Scrophularia species leading to strong growth inhibition and high cell death rates. The expression of cell cycle regulators, oncogenes and cell death inducers was determined by Western blot analysis. Furthermore the effect of S. lucida was studied in an NF-κB reporter assay, and in a novel assay measuring 'circular chemo-repellent-induced defects' (CCID) in lymph endothelial monolayers that were induced by MCF-7 breast cancer spheroids. Methanol extracts of Scrophularia species exhibited strong anti-proliferative properties. S. floribunda extract inhibited G2/M- and later on S-phase and S. lucida inhibited S-phase and in both cases this was associated with the down-regulation of c-Myc expression. Extracts of S. floribunda and S. lucida led to necrosis and apoptosis, respectively. Furthermore, S. lucida, but not S. floribunda, effectively attenuated tumour cell intravasation through lymph endothelial cell monolayers, which correlated with the inhibition of NF-κB. S. lucida exhibited promising anti-neoplastic effects and this was most likely due to the down-regulation of various cell cycle regulators, proto-oncogenes and NF-κB and the activation of caspase-3.
... For example, ERK contributes to the induction of neuronal differentiation by nerve growth factor (29) and to growth arrest and the induction of apoptosis through phosphoactivation of p53 (30,31). Elsewhere, several studies have demonstrated that ERK signaling is associated with up-regulation of p21 expression in a variety of cell types (32)(33)(34)(35)(36)(37)(38). However, despite the emerging recognition that MAPKs inhibit cell proliferation by affecting p21 expression, little is yet known about the mechanisms by which these kinases regulate p21 transcription. ...
Cyclin-dependent kinase inhibitor (CDKN1A), often referred to as p21Waf1/Cip1 (p21), is induced by a variety of environmental stresses. Transcription factor ELK-1 is a member of the ETS oncogene superfamily.
Here, we show that ELK-1 directly trans-activates the p21 gene, independently of p53 and EGR-1, in sodium arsenite (NaASO2)-exposed HaCaT cells. Promoter deletion analysis and site-directed mutagenesis identified the presence of an ELK-1-binding
core motif between −190 and −170 bp of the p21 promoter that confers inducibility by NaASO2. Chromatin immunoprecipitation and electrophoretic mobility shift analyses confirmed the specific binding of ELK-1 to its
putative binding sequence within the p21 promoter. In addition, NaASO2-induced p21 promoter activity was enhanced by exogenous expression of ELK-1 and reduced by expression of siRNA targeted to ELK-1 mRNA.
The importance of ELK-1 in response to NaASO2 was further confirmed by the observation that stable expression of ELK-1 siRNA in HaCaT cells resulted in the attenuation
of NaASO2-induced p21 expression. Although ELK-1 was activated by ERK, JNK, and p38 MAPK in response to NaASO2, ELK-1-mediated activation of the p21 promoter was largely dependent on ERK. In addition, EGR-1 induced by ELK-1 seemed to be involved in NaASO2-induced expression of BAX. This supports the view that the ERK/ELK-1 cascade is involved in p53-independent induction of
p21 and BAX gene expression.
... Previous studies have shown that UCN-01 and Gö6976 sensitized cells to gemcitabine via Chk1 inhibition, resulting in abrogation of the cell cycle arrest and subsequent cell death. (36)(37)(38)(39) We examined the sensitivity of Chk1 signalinginhibited cells to gemcitabine under the hypoglycemic ⁄ hypoxic condition. Western blot analysis showed that 1 lM of the Chk1 inhibitors, UCN-01 and Gö6976, reduced the phosphorylation of cdc25c, a downstream mediator of Chk1 (Fig. 3A); UCN-01 and Gö6976 lowered the IC 50 of gemcitabine by more than 10 times under the normoglycemic ⁄ normoxic condition, but not under the hypoglycemic ⁄ hypoxic condition (Fig. 3B,C). ...
Tumor tissues are often hypoxic because of defective vasculature. We previously showed that tumor tissues are also often deprived of glucose. The efficacy of anticancer drugs is affected by the tumor microenvironment, partly because of the drug delivery and cellular drug resistance; however, the precise mechanisms remain to be clarified. In the present study, we attempted to clarify whether hypoglycemic/hypoxic condition, which mimics the tumor microenvironment, might induce drug resistance, and if it did, to elucidate the underlying mechanisms. Pancreatic cancer-derived PANC-1 cells were treated with serial dilutions of anticancer drugs and incubated in either normoglycemic (1.0 g/L glucose) or hypoglycemic (0 g/L glucose) and normoxic (21% O(2)) or hypoxic (1% O(2) ) conditions. The 50% inhibitory concentration of gemcitabine was 1000 times higher for PANC-1 cells incubated under the hypoglycemic/hypoxic condition than for those incubated under the normoglycemic/normoxic condition. Conventional anticancer drugs target rapidly growing cells, so that non-proliferating or slowly proliferating cells usually show resistance to drugs. Though the cell cycle was delayed, sufficient cellular uptake and DNA incorporation of gemcitabine occurred under the hypoglycemic/hypoxic condition to cause DNA lesions and S-phase arrest. To overcome hypoglycemic/hypoxia-induced drug resistance, we examined kinase inhibitors targeting Chk1 or cell-survival signaling pathways. Among the compounds examined, the combination of UCN-01 and LY294002 partially sensitized the cells to gemcitabine under the hypoglycemic/hypoxic condition. These findings suggested that the adoption of suitable strategies may enhance the cytotoxicities of clinically used anticancer drugs against cancer cells.
