NDF/heregulin-induced cell cycle changes and apoptosis in breast tumour cells: role of PI3 kinase and p38 MAP kinase pathways.

Neu differentiation factor (NDF)/heregulin activates ErbB2 via heterodimerization with the NDF receptors ErbB3 and ErbB4. Cells which express normal levels of these receptors are often growth stimulated by NDF, whereas SKBR3, and other ErbB2-overexpressing breast tumour cells are growth inhibited. We demonstrate here that in SKBR3 cells, NDF induces G1 progression but also causes a G2 delay from day 1 and apoptosis from days 2-3. G1 progression was associated with ErbB2 transactivation of ErbB3 and subsequent stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway whereas apoptosis was dependent on p38 MAPK. Inhibition of ERK1/ERK2 had no effect on cell cycle progression or apoptosis. Activation of ErbB3 and PI3K was also seen with betacellulin (BTC) but not epidermal growth factor (EGF) and correlated with the growth effects of these ligands. All three ligands induced short-term activation of p38 MAPK in a c-Src-dependent manner. However, only NDF caused a second, c-Src-independent increase in p38 MAPK activity which was required for apoptosis.     

Role of p53 and p38 MAP kinase in nitric oxide-induced G2/M arrest and apoptosis in the human lung carcinoma cells

The authors, Jui-I.Chao and Pao-Chen Kuo, found critical problemswith some of the data they reported in the above paper and wishto withdraw it from publication. The paper has been publishedonline     

Human p14(ARF)-mediated cell cycle arrest strictly depends on intact p53 signaling pathways

The tumor suppressor ARF is transcribed from the INK4a/ARF locus in partly overlapping reading frames with the CDK inhibitor p16(Ink4a). ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19(ARF). Little is known about the dependency of human p14(ARF) function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14(ARF)-induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53(+/+)p21(CIP1+/+) 14-3-3sigma(+/+)), but not p53(-/-) counterparts, underwent G(1) and G(2) cell cycle arrest following infection with a p14(ARF)-adenovirus. In p21(CIP1-/-) cells, p14(ARF) did not induce G(1) or G(2) arrest, while 14-3-3sigma(-/-) counterparts were mainly arrested in G(1), pointing to essential roles of p21(CIP1) in G(1) and G(2) arrest and cooperative roles of p21 and 14-3-3sigma in ARF-mediated G(2) arrest. Our data demonstrate a strict p53 and p21(CIP1) dependency of p14(ARF)-induced cell cycle arrest in human cells.     

Clusterin-mediated apoptosis is regulated by adenomatous polyposis coli and is p21 dependent but p53 independent

Clusterin is a widely expressed glycoprotein that has been paradoxically observed to have both pro- and antiapoptotic functions. Recent reports suggest this apparent dichotomy of function may be related to two different isoforms, one secreted and cytoplasmic, the other nuclear. To clarify the functional role of clusterin in regulating apoptosis, we examined its expression in human colon cancer tissues and in human colon cancer cell lines. We additionally explored its expression and activity using models of adenomatous polyposis coli (APC)- and chemotherapy-induced apoptosis. Clusterin RNA and protein levels were decreased in colon cancer tissues largely devoid of wild-type APC when compared with matched normal tissue controls, suggesting a means for invasive cancers to avoid apoptosis. Conversely, induction of apoptosis by expression of wild-type APC or by treatment with chemotherapy led to increased clusterin RNA and protein levels localizing to apoptotic nuclei. We found that transient transfection of clusterin to colon cancer cell lines directly enhanced basal and chemotherapy-induced apoptosis. Clusterin-induced apoptosis was inhibited by antisense clusterin and was found to be highly dependent on p21 but not p53 expression, yet a deficit in p21 can be subverted by clusterin transfection. Collectively, these data support the hypothesis that nuclear clusterin function is proapoptotic when induced by APC or chemotherapy in the context of p21 expression. Absent of p21, clusterin in not induced, and apoptosis is significantly inhibited. These data support a potential therapeutic role for clusterin in enhancing chemotherapy-induced apoptosis and in promoting apoptosis in cells deficient in p21.     

Flavopiridol induces apoptosis in B-cell chronic lymphocytic leukaemia cells through a p38 and ERK MAP kinase-dependent mechanism

Flavopiridol, a synthetic flavone, has been previously shown to induce apoptosis in B-cell chronic lymphocytic leukaemia (B-CLL) cells in vitro. The apoptosis was associated with a concomitant activation of caspase-3 without evidence of dependence on functional p53 or Bcl-2 family modulation. In this study, we examined flavopiridol-induced apoptosis in terms of upstream caspase activity, cell cycle distribution and signal transduction, in order to elucidate the mechanism of action of this potent cytotoxic agent. Flavopiridol-induced apoptosis was significantly abrogated by the caspase-9 inhibitor Z-LEHD-FMK (p = 0.002; paired t-test) but was not altered by the caspase-8 inhibitor Z-IETD-FMK (p = 0.37; paired t-test). There was a concentration-dependent increase in a sub G0/G1 peak indicative of apoptotic cells but if these cells were excluded by gating no other cell cycle perturbations were observed suggesting that flavopiridol is capable of inducing apoptosis in cells in all phases of the cell cycle. Significantly, apoptosis was associated with activation of p38 MAP kinase and suppression of ERK activity (p = 0.0036 and p = 0.0048, respectively; paired t-test). These results show for the first time that flavopiridol modulates specific cellular signal transduction pathways in B-CLL cells thereby altering the balance between survival and cell death signals and providing a rationale for the p53-independent nature of flavopiridol-induced apoptosis. Further work is required to identify whether combinations of conventional chemotherapeutic drugs and novel agents like flavopiridol can be used to improve patient outcomes in the treatment of B-CLL.     

Attenuation of BPDE-induced p53 accumulation by TPA is associated with a decrease in stability and phosphorylation of p53 and downregulation of NFkappaB activation: role of p38 MAP kinase

DNA damage caused by benzo[a]pyrene (B[a]P) or other polynuclear hydrocarbons (PAHs) induce p53 protein as a protective measure to eliminate the possibility of mutagenic fixation of the DNA damage. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits p53 response induced by B[a]P and other DNA-damaging agents and may cause tumor promotion. The molecular mechanism of attenuation of B[a]P-induced p53 response by TPA is not known. We investigated the effect of TPA on p53 response in (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-treated mouse epidermal JB6(P(+)) Cl 41 cells. BPDE treatment induced p53 accumulation which was attenuated significantly by TPA. Cells treated with BPDE and TPA showed increased ratio of Mdm2 to p53 proteins in p53 immunoprecipitate and decreased p53 life span compared to BPDE-treated cells indicating p53 destabilization by TPA. TPA also inhibited BPDE-induced p53 phosphorylation at serine15. Activation of both ERKs and p38 MAPK by BPDE and attenuation of BPDE-induced p53 accumulation by U0126 or SB202190, specific inhibitor of MEK1/2 or p38 MAPK, indicate the role of ERKs and p38 MAPK in p53 accumulation. Interestingly, TPA potentiated BPDE-induced activation of ERKs whereas p38 MAPK activation was significantly inhibited by TPA, suggesting that inhibition of p38 MAPK is involved in p53 attenuation by TPA. Furthermore, SB202190 treatment caused decreased p53 stability and inhibition of phosphorylation of p53 at serine15 in BPDE-treated cells. We also observed that TPA or SB202190 attenuated BPDE-induced nuclear factor kappa B (NFkappaB) activation in JB6 Cl 41 cells harboring NFkappaB reporter plasmid. To our knowledge this is the first report that TPA inhibits chemical carcinogen-induced NFkappaB activation. Interference of TPA with BPDE-induced NFkappaB activation implicates abrogation of p53 function which has been discussed. Overall, our data suggest that abrogation of BPDE-induced p53 response and of NFkappaB activation by TPA is mediated by impairment of the signaling pathway involving p38 MAPK.     

Differential regulation of Jun N-terminal kinase and p38MAP kinase by Galpha12

Based on the findings that the overexpression of the wild-type Galpha(12) (Galpha(12)WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Galpha(12) can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Galpha(12)WT or the expression of an activated mutant of Galpha(12) (Galpha(12)QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Galpha(12)WT or the expression of Galpha(12)QL results in a potent inhibition of p38MAPK, and that the mechanism by which Galpha(12) inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Galpha(12) attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Galpha(12) activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Galpha(12) differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Galpha(12) stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways.     

Mutant p53 is constitutively phosphorylated at Serine 15 in UV-induced mouse skin tumors: involvement of ERK1/2 MAP kinase

Upon DNA damage, phosphorylation and nuclear translocation of wild-type p53 tumor suppressor protein signals its functional activation. However, very little is known about phosphorylation and localization of mutant p53. We found that mutant p53 protein in UV-induced murine primary skin tumors and cultured cell lines was constitutively phosphorylated at serine 15 residue and localized in the cell's nuclei. To investigate the mechanism of constitutive phosphorylation of mutant p53, we tested the involvement of a wide range of protein kinases and found that ERK1/2 mitogen-activated protein kinase was physically associated with mutant p53 in the nucleus. Addition of active recombinant ERK2 kinase protein in vitro to immunoprecipitated mutant p53 resulted in increased phosphorylation at serine 15. Furthermore, ERK1/2 activity was higher in tumor cells than normal cells, suggesting that phosphorylation of mutant p53 at serine 15 depends on the level of ERK1/2 activation. Interestingly, accumulation of mutant p53 in tumor cells was paralleled by low levels of Murine Double Minute 2 protein (MDM2) expression. However, when MDM2 was overexpressed, the fraction of mutant p53 that was phosphorylated at serine 15 resisted degradation, whereas the level of total p53 decreased, suggesting that phosphorylation at serine 15 and downregulation of MDM2 protein may both contribute to stabilization of mutant p53 in tumor cells.     

Roles of p38- and c-jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiol-induced p53 induction and apoptosis

As 2-methoxyestradiol (2-ME), an endogenous estrogen metabolite, has been established to cause apoptosis of prostate cancer cells, the downstream effectors of the signaling remain unclear. In the current study, we investigated molecular mechanisms by which 2-ME induces apoptosis in human prostate cancer cell line, LNCaP. It was found that 2-ME mediates apoptosis through p53 induction. Nuclear factor kappaB (NFkappaB) was activated by 2-ME and closely regulated by the mitogen-activated protein kinase, p38. Inhibition of p38 or NFkappaB resulted in suppression of p53 induction and apoptosis. Moreover, we demonstrated that 2-ME activates the c-jun NH2-terminal kinase (JNK)/activation protein (AP)-1 pathway. Interestingly, inhibition of JNK strongly reduced Bcl-2 phosphorylation by 2-ME as well as p53 induction, and almost completely suppressed 2-ME-induced apoptosis. Androgen stimulation with dihydrotestosterone, a major endogenous metabolite of testosterone, also significantly inhibited p38/NFkappaB and JNK/AP-1 activation and apoptosis. The results suggest that not only p53 induction through p38/JNK-dependent NFkappaB/AP-1 activation but also JNK-dependent Bcl-2 phosphorylation are required for 2-ME-induced apoptosis; moreover, inhibition of these pathways may be involved in androgen-mediated resistance to apoptosis.     

p38MAPK在EGCG诱导人胃癌MGC803细胞凋亡中的作用

目的：探讨p38丝裂原活化蛋白激酶（p38 mitogen—activaced protein kinase，v38MAPK）在表没食子儿茶素没食子酸酯（epigallocatechin-3-gallate，EGCG）诱导人胃癌MGC803细胞凋亡中的作用。方法：采用MTT法检测MGC803细胞的存活率，荧光显微镜观察和碘化丙啶染色FCM检测MGC803细胞凋亡率，Western印迹法检测MGC803细胞中p38MAPK蛋白及磷酸化p38MAPK蛋白的表达。结果：EGCG可诱导MGC803细胞凋亡，且p38MAPK被激活。用p38MAPK特异性抑制剂SB203580干预后，EGCG抑制MGC803细胞生长的作用明显减弱，细胞凋亡率下降，p38MAPK活性显著下降。结论：EGCG可诱导MGC803细胞凋亡，该作用可被SB203580显著抑制，提示EGCG可能通过激活D38MAPK使部分MGCR03细胞凋亡。     

Expression of the novel tumour suppressor p33(ING1)is independent of p53

A recently cloned tumour suppressor candidate, p33ING1, has been shown in vitro to collaborate with p53 to execute growth arrest and apoptosis. However, it is unclear as to how the expression of ING1 is regulated in normal and stress conditions. Using a p53-knockout mouse model, we investigated if the expression of ING1 was dependent on p53. We found that there was no difference in ING1 mRNA and protein levels between p53+/+ and p53-/- murine organs. In addition, when normal human epithelial keratinocytes (NHEK) and a keratinocyte cell line, HaCaT, which lacks wild-type p53 function, were exposed to UVB irradiation, the expression levels of ING1 were elevated in both NHEK and HaCaT cells. It is interesting, however, that UVB irradiation did not induce ING1 expression in dermal fibroblasts isolated from p53+/+ and p53-/- mice. Based on our findings, we therefore conclude that the expression of ING1 is independent of p53 status. UV induction of ING1 in keratinocytes suggests that ING1 may play a role in cellular stress response and skin carcinogenesis.     

A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation.

In the present study we examined in more detail the dual role of the c-JUN N-terminal kinase (JNK) and p38 stress-activated protein kinase pathways in mediating apoptosis or cellular activation in hematopoietic cells. Growth factor deprivation of the erythroleukemic cell line TF-1 led to apoptosis which was associated with an enhanced activity of JNK and p38 and immediate dephosphorylation of the extracellular signal-regulated kinases (ERKs). Enhanced activity of p38 and JNK was not only observed during apoptosis but also in TF-1 cells stimulated with IL-1. IL-1 rescued TF-1 cells from apoptosis. In this case, the upregulation of p38 and JNK was associated with an enhanced activity of ERK. By using SB203580, a specific inhibitor of the p38 signaling pathway, it was demonstrated that p38 plays a pivotal role in the apoptotic process. SB203580 repressed the apoptotic process to a large extent. In contrast, PD98059, a specific inhibitor of the ERK pathway, counteracted the suppressive effects of SB203580 and IL-1 on the apoptotic process indicating that the protective effect of SB203580 and IL-1 might be the result of a shift in the balance between the ERK1/2 and p38/JNK route. This was also supported by experiments with TF-1 cells overexpressing the Shc protein that demonstrated a significantly lower percentage of apoptotic cells, which coincided with higher ERK activity. Finally, the IL-1 and SB203580-mediated effects were associated with an enhanced nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activity, which could also be blocked by PD98059. These data demonstrate a dual function of the p38 pathway whereby other factors, such as ERK kinases, AP-1 and NF-kappaB, might determine the final cellular response.     

Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status

The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.