Dominant-negative P53 Mutant Research Articles

Identification of an ataxia telangiectasia-mutated protein mediated surveillance system to regulate Bcl-2 overexpression

Bcl-2 can both promote and attenuate tumorigenesis. Although the former function is relatively well characterized, the mechanism of the latter remains elusive. We report here that enforced Bcl-2 expression in MCF7 cells stabilizes p53, induces phosphorylation of p53 serine 15 (p53pSer15) and inhibits MCF7 cell growth. Consistent with p53 Ser15 being a target of ataxia telangiectasia mutated protein(ATM)/ATR (ATM- and rad3-related) in the DNA damage response, Bcl-2 activates ATM by inducing ATM Ser1981 phosphorylation, which is accompanied with the phosphorylaton of two additional ATM substrates, Chk2 Thr68 and H2AX Ser139. Downregulation of ATM using a specific small interference RNA fragment (ATMRNAi) abolished Bcl-2-induced p53pSer15 and Bcl-2-mediated growth inhibition of MCF7 cells. Ectopic expression of a dominant-negative p53 mutant, p53175H, partially rescued this growth inhibition. Taken together, these observations demonstrate the contribution of ATM-p53 function to Bcl-2-mediated inhibition of MCF7 cell growth, indicating an ATM-mediated surveillance system for regulating Bcl-2 overexpression. Consistent with this concept, we found that MCF7 cells express Bcl-2 heterogeneously with 34.5% of cells being Bcl-2 negative. In general, Bcl-2-positive MCF7 cells proliferate slower than those of Bcl-2 negative. Thus, we provide evidence suggesting that activation of ATM suppresses Bcl-2-induced tumorigenesis, and that attenuation of ATM function may be an important event in breast cancer progression.

Selective Requirement of p38α MAPK in Cytokine-Dependent, but Not Antigen Receptor-Dependent, Th1 Responses

The role of the p38 MAPK pathway in Th1 development has been controversial, because indirect manipulations of either upstream p38 activators or modifiers of p38 activity have had variable effects on IFN-gamma production in CD4+ T cells. Uncertainties regarding the specificity of pharmacologic inhibition or p38 dominant negative mutants diminish the strength of conclusions about the role of the p38alpha isoform in Th1 development. Also, the effects of some upstream p38 activators, such as MAPK kinase 3, on Th1 development are not as strong as the effects of other manipulations, such as the expression of a dominant negative p38 mutant. Finally, embryonic lethality has prevented a direct examination of p38alpha-deficient T cells. To test the requirement for p38alpha in Th1 differentiation, we generated Ag-specific p38alpha+/- and p38alpha-/- CD4+ T cells using RAG2-/- blastocyst complementation and retroviral expression of the DO11.10 TCR. IFN-gamma production in response to TCR signaling is normal in p38alpha-/- T cells cultured in Th1 conditions, implying normal Th1 development. However, p38alpha-/- Th1 cells have a much greater defect in IFN-gamma secretion stimulated by IL-12/IL-18 compared with TCR-induced IFN-gamma secretion. These results suggest that the activity of p38alpha in Th1 cells is relatively restricted to acting in one of two alternative pathways (i.e., cytokine induced) that can induce the production of IFN-gamma in differentiated Th1 cells, but that p38alpha is not required for the process of Th1 commitment and development itself.

Dicoumarol potentiates cisplatin-induced apoptosis mediated by c-Jun N-terminal kinase in p53 wild-type urogenital cancer cell lines

3-3'-Methylene-bis [4-hydroxycoumarin] (dicoumarol), an inhibitor of NADPH:quinone oxidoreductase 1, has been reported to possess potential antineoplastic effects and the ability to abrogate p53 protein. In the present study, we investigated the cytotoxic effects of dicoumarol in combination with cisplatin (CDDP), using four bladder (RT112, 253J, J82 and UMUC3) and two prostate (LNCap and PC3) cancer cell lines. Single treatment with 100 microM dicoumarol suppressed cell proliferation but did not induce apoptosis at 24 h in all cell lines examined. On the other hand, pretreatment with dicoumarol enhanced cytotoxicity of CDDP in three cell lines with wild type of p53 (RT112, 253J and LNCap), but not in three other cell lines with mutant p53 or in RT112 stable transfectants with a dominant-negative mutant of p53. In RT112 and LNCap, CDDP induced p53 and p21 expression, while pretreatment of dicoumarol suppressed induction of p53/p21 and resulted in sequential activation of c-Jun N-terminal kinase (JNK) in a time-dependent manner. Furthermore, inhibition of JNK, using SP600125, completely suppressed activity of caspases and poly-(ADP-ribose) polymerase cleavage, leading to suppression of enhancement of CDDP-mediated apoptosis by dicoumarol. These results suggested that dicoumarol could enhance cytotoxicity of CDDP in urogenital cancer cells with wild-type p53 through the p53/p21/JNK pathways.

Estrogen Prevents Cardiomyocyte Apoptosis through Inhibition of Reactive Oxygen Species and Differential Regulation of p38 Kinase Isoforms

From human and animal studies, estrogen is known to protect the myocardium from an ischemic insult. However, there is limited knowledge regarding mechanisms by which estrogen directly protects cardiomyocytes. In this report, we employed an in vitro model, in which cultured rat cardiomyocytes underwent prolonged hypoxia followed by reoxygenation (H/R), to study the cardioprotective mechanism of estrogen. 17-beta-estradiol (E2) acting via estrogen receptors inhibited H/R-induced apoptosis of cardiomyocytes. Mitochondrial reactive oxygen species (ROS) generated from H/R activated p38alpha MAPK, and inhibition of p38alpha with SB203580 significantly prevented H/R-induced cell death. E2 suppressed ROS formation and p38alpha activation by H/R and concomitantly augmented the activity of p38beta. Unlike p38alpha, p38beta was little affected by H/R. Dominant negative p38beta protein expression decreased E2-mediated cardiomyocyte survival and ROS suppression during H/R stress. The prosurvival signaling molecule, phosphoinositol-3 kinase (PI3K), has previously been linked to cell survival following ischemia-reperfusion injury. Here, E2-activated PI3K was found to inhibit ROS generated from H/R injury, leading to inhibition of downstream p38alpha. We further linked these signaling pathways in that p38beta was activated by E2 stimulation of PI3K. Thus, E2 differentially modulated two major isoforms of p38, leading to cardiomyocyte survival. This was achieved by signaling through PI3K, integrating cell survival mediators.

Sequential activation of p38 and ERK pathways by cGMP-dependent protein kinase leading to activation of the platelet integrin αIIbβ3

AbstractIntegrin activation (inside-out signaling) in platelets can be initiated by agonists such as von Willebrand factor (VWF) and thrombin. Here we show that a mitogen-activated protein kinase (MAPK), p38, plays an important role in the activation of integrin αIIbβ3 induced by VWF and thrombin. A dominant-negative mutant of p38, p38AF, inhibits αIIbβ3 activation induced by VWF binding to its receptor, the platelet glycoprotein Ib-IX (GPIb-IX), and p38 inhibitors diminish platelet aggregation induced by VWF or low-dose thrombin. The inhibitory effect of p38 inhibitor is unlikely to be caused by the previous suggested effect on cyclo-oxygenase, as inhibition also was observed in the presence of high concentrations of cyclo-oxygenase inhibitor, aspirin. VWF or thrombin induces p38 activation, which is inhibited in cGMP-dependent protein kinase (PKG)-knockout mouse platelets and PKG inhibitor-treated human platelets, indicating that activation of p38 is downstream from PKG in the signaling pathway. p38AF or p38 inhibitors diminish PKG-induced phosphorylation of extracellular stimuli-responsive kinase (ERK), which also is important in integrin activation. Thus, p38 plays an important role in mediating PKG-dependent activation of ERK. These data delineate a novel signaling pathway in which platelet agonists sequentially activate PKG, p38, and ERK pathways leading to integrin activation.

PUMA-mediated apoptosis in fibroblast-like synoviocytes does not require p53

PUMA (p53-upregulated modulator of apoptosis) is a pro-apoptotic gene that can induce rapid cell death through a p53-dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53-/- murine FLS were also examined to evaluate whether p53 is required. p53-deficient or control FLS were transfected with PUMA cDNA or empty vector. p53 and p21 expression were then determined by Western blot analysis. Apoptosis was assayed by ELISA to measure histone release and caspase-3 activation, or by trypan blue dye exclusion to measure cell viability. Initial studies showed that p53 siRNA decreased p53 expression by more than 98% in human FLS. Loss of p53 increased the growth rate of cells and suppressed p21 expression. However, PUMA still induced apoptosis in control and p53-deficient FLS after PUMA cDNA transfection. Similar results were observed in p53-/- murine FLS or in human FLS transfected with a dominant-negative mutant p53 gene. These data suggest that PUMA-induced apoptosis in FLS does not require p53. Therefore, approaches to gene therapy that involve increasing PUMA expression could be an effective inducer of synoviocyte cell death in rheumatoid arthritis regardless of the p53 status in the synovium.

The p38 mitogen‐activated protein kinase regulates effector functions of primary human CD4 T cells

The role of p38 mitogen-activated protein kinase in primary human T cells is incompletely understood. We analyzed in detail the role of p38 in the regulation of effector functions and differentiation of human CD4 T cells by using a p38-specific inhibitor and a dominant-negative mutant of p38. p38 was found to mediate expression of IL-10 and the Th2 cytokines IL-4, IL-5, and IL-13 in both, primary naive and memory T cells. In contrast, inhibition of p38 activity did not affect expression of the Th1 cytokines IFN-gamma and TNF induced by TCR-stimulation, but decreased IL-12-mediated IFN-gamma expression. Cytokine expression from established Th2 effector cells was also regulated by p38, however, the role of p38 was less pronounced compared to primary CD4 T cells. p38 MAPK regulated cytokine gene expression at both, the transcriptional level by activating gene transcription and the post-transcriptional level by stabilizing cytokine mRNA. As a result of the effect of p38 on IL-4 expression, p38 activity modulated differentiation of naive precursor T cells by inducing a shift of the Th1/Th2 balance toward the immuno-modulatory Th2 direction. Together, the data suggest that p38 plays a key role in human Th2 cell immune responses.

Role of the mismatch repair system and p53 in the clastogenicity and cytotoxicity induced by bleomycin

The mismatch repair (MMR) system and p53 protein play a pivotal role in maintaining genomic stability and modulate cell chemosensitivity. Aim of this study was to examine the effects of either MMR-deficiency or p53 inactivation, or both, on cellular responses to bleomycin. The MMR-deficient colon carcinoma cell line HCT116 and its MMR-proficient subline HCT116/3–6, both expressing wild-type p53, were transfected with an expression vector encoding a dominant-negative p53 mutant, or with the empty vector. Four transfected clones, having the following phenotypes, MMR-proficient/p53 wild-type, MMR-proficient/p53 mutant, MMR-deficient/p53 wild-type, MMR-deficient/p53 mutant, were subjected to treatment with bleomycin. Loss of MMR function alone was associated with increased resistance to apoptosis, chromosomal damage and inhibition of colony formation caused by bleomycin. Loss of p53 alone resulted in abrogation of G 1 arrest and increased sensitivity to apoptosis and chromosomal damage induced by the drug, but did not affect clonogenic survival after bleomycin treatment. Disabling both p53 and MMR function led to abrogation of G 1 arrest and to a moderate impairment of drug-induced apoptosis. Chromosomal damage was reduced in the MMR-deficient/p53 mutant clone with respect to the MMR-proficient/p53 wild-type one, when evaluated 48 h after bleomycin treatment, but was comparable in both clones 96 h after drug exposure. Clonogenic survival of the MMR-deficient/p53 mutant clone was similar to that of the MMR-deficient/p53 wild-type one. The effects of MMR-deficiency on cellular responses to bleomycin were confirmed using the MMR-proficient lymphoblastoid cell line TK6 and its MMR-deficient subline MT1, both expressing wild-type p53. In conclusion, our data show that loss of MMR and p53 function exerts opposite and independent effects on apoptosis and chromosomal damage induced by bleomycin. Moreover, inactivation of MMR confers resistance to the cytotoxic activity of the anticancer agent in cells expressing either wild-type or mutant p53.

Functional analysis of p53 gene and the prognostic impact of dominant‐negative p53 mutation in endometrial cancer

In addition to the loss of function, mutant p53 can possess a dominant-negative effect on wild-type p53 and may also exert gain-of-function activity. It is not clear whether the functional status of p53 mutation contributes to differences in outcome in endometrial cancer. We collected a total of 92 RNA samples of high quality from endometrial cancer tissues, and the samples were subjected to yeast functional assay and sequencing for p53 mutations. The detected mutant p53 genes were further investigated for their dominant-negative activity using a yeast-based transdominance assay. p53 mutation was found in 24 out of 92 (26.1%) tumors, of which 10 exhibited no dominant-negative activity (recessive mutation) and 14 showed dominant-negative activity. Dominant-negative p53 mutation was related to advanced stages (p = 0.01), nonendometrioid type tumors (p = 0.01) and grade 3 tumors (p = 0.04). The patients with dominant-negative mutation had significantly shorter survival than patients with no mutation (p < 0.0001) and those with a recessive mutation (p = 0.01) in the p53 gene. No difference in survival was found between the patients with tumors harboring a recessive p53 mutation and those with tumors harboring a wild-type p53. Multivariate analysis revealed that dominant-negative p53 mutation (p = 0.019), FIGO stage (p = 0.0037) and histologic subtype (p = 0.014) were independently related to patient survival. Dominant-negative p53 mutation was the most important prognostic factor for stage III/IV endometrial cancer (p = 0.0023). In conclusion, dominant-negative p53 mutation is often found in advanced stages and aggressive histologic subtypes of endometrial cancer and it is a strong predictor of survival of patients with advanced endometrial cancer. To elucidate further the role of p53 mutation in endometrial cancer, it is necessary to investigate gain-of-function activity involving dominant-negative p53 mutant proteins.

Characterization of the Signaling Pathway Downstream p75 Neurotrophin Receptor Involved in β-Amyloid Peptide-Dependent Cell Death

The accumulation of beta-amyloid (Abeta) peptide is a key pathogenic event in Alzheimer's disease. Previous studies have shown that Abeta peptide can damage neurons by activating the p75 neurotrophin receptor (p75NTR). However, the signaling pathway leading to neuronal cell death is not completely understood. By using a neuroblastoma cell line devoid of neurotrophin receptors and engineered to express either a full-length or a death domain (DD)-truncated form of p75NTR, we demonstrated that Abeta peptide activates the mitogen-activated protein kinases (MAPKs) p38 and c-Jun N-terminal kinase (JNK). We also found that Abeta peptide induces the translocation of nuclear factor-kappaB (NF-kappaB). These events depend on the DD of p75NTR. Beta-amyloid (Abeta) peptide was found not to be toxic when the above interactors were inhibited, indicating that they are required for Abeta-induced neuronal cell death. p75 neurotrophin receptor (p75NTR)-expressing cells became resistant to Abeta toxicity when transfected with dominant-negative mutants of MAPK kinases 3, 4, or 6 (MKK3, MKK4, or MKK6), the inhibitor of kappaBalpha, or when treated with chemical inhibitors of p38 and JNK. Furthermore, p75NTR-expressing cells became resistant to Abeta peptide upon transfection with a dominant-negative mutant of p53. These results were obtained in the presence of normal p38 and JNK activation, indicating that p53 acts downstream of p38 and JNK. Finally, we demonstrated that NF-kappaB activation is dependent on p38 and JNK activation. Therefore, our data suggest a signaling pathway in which Abeta peptide binds to p75NTR and activates p38 and JNK in a DD-dependent manner, followed by NF-kappaB translocation and p53 activation.

Histone H3 acetylation is associated with reduced p21WAF1/CIP1 expression by gastric carcinoma

Histone acetylation appears to play an important role in transcriptional regulation. Inactivation of chromatin by histone deacetylation is involved in the transcriptional repression of several tumour suppressor genes, including p21(WAF1/CIP1). However, the in vivo status of histone acetylation in human cancers, including gastric carcinoma, is not well understood. This study shows that histone H3 in the p21(WAF1/CIP1) promoter region is hypoacetylated and that this hypoacetylation is associated with reduced p21(WAF1/CIP1) expression in gastric carcinoma specimens. Chromatin immunoprecipitation assays revealed that histone H3 was hypoacetylated in the p21(WAF1/CIP1) promoter and coding regions in 10 (34.5%) and 10 (34.5%) of 29 gastric carcinoma specimens, respectively. Hypoacetylation of histone H4 in the p21(WAF1/CIP1) promoter and coding regions was observed in 6 (20.7%) and 16 (55.2%) of 29 gastric carcinoma specimens, respectively. p21(WAF1/CIP1) mRNA levels were associated with histone H3 acetylation status in the p21(WAF1/CIP1) promoter region (p = 0.047) but not p53 mutation status (p = 0.460). In gastric carcinoma cell lines, expression of p21(WAF1/CIP1) protein was induced by trichostatin A, a histone deacetylase inhibitor. This induction was associated with hyperacetylation of histone H3 in the p21(WAF1/CIP1) promoter region. Hyperacetylation of histone H4 in the p21(WAF1/CIP1) promoter region did not appear to be associated with increased expression. Induction of p21(WAF1/CIP1) protein expression was associated with hyperacetylation of histones H3 and H4 in the p21(WAF1/CIP1) coding region. Expression of a dominant-negative mutant of p53 reduced expression of p21(WAF1/CIP1) protein. Histone H4 acetylation in both the promoter and coding regions of the p21(WAF1/CIP1) gene in cells expressing dominant-negative p53 was less than half of that in cells expressing wild-type p53, whereas histone H3 acetylation in both the promoter and coding regions was slightly reduced (by approximately 20%) in cells expressing the dominant-negative p53. These findings provide evidence that alteration of histone acetylation occurs in human cancer tissue specimens such as those from gastric carcinoma.

Role of p53 in cisplatin-induced tubular cell apoptosis: dependence on p53 transcriptional activity.

Tubular damage by cisplatin leads to acute renal failure, which limits its use in cancer therapy. In tubular cells, a primary target for cisplatin is presumably the genomic DNA. However, the pathway relaying the signals of DNA damage to tubular cell death is unclear. In response to DNA damage, the tumor suppressor gene p53 is induced and is implicated in subsequent DNA repair and cell death by apoptosis. The current study was designed to examine the role of p53 in cisplatin-induced apoptosis in cultured rat kidney proximal tubular cells. Cisplatin at 20 microM induced apoptosis in approximately 70% of cells, which was partially suppressed by carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone (VAD), a general caspase inhibitor. Of interest, cisplatin-induced apoptosis was also suppressed by pifithrin-alpha, a pharmacological inhibitor of p53. Cisplatin-induced caspase activation was completely inhibited by VAD, but only partially by pifithrin-alpha. Early during cisplatin treatment, p53 was phosphorylated and upregulated. The p53 activation was blocked by pifithrin-alpha, but not by VAD. Bcl-2 expression abolished cisplatin-induced apoptosis without blocking p53 phosphorylation or induction. The results suggest that p53 activation might be an early signal for apoptosis during cisplatin treatment. To further determine the role of p53, tubular cells were stably transfected with a dominant-negative mutant of p53 with diminished transcriptional activity. Expression of the mutant attenuated cisplatin-induced apoptosis and caspase activation. In conclusion, the results support an important role for p53 in cisplatin-induced apoptosis in renal tubular cells. p53 May regulate apoptosis through the transcription of apoptotic genes.

Ha-Ras(G12V) induces senescence in primary and immortalized human esophageal keratinocytes with p53 dysfunction.

Oncogenic Ras induces premature senescence in primary cells. Such an oncogene-induced senescence involves activation of tumor suppressor genes that provide a checkpoint mechanism against malignant transformation. In mouse, the ARF-p53 pathway mediates Ha-Ras(G12V)-induced senescence, and p19(ARF-/-) and p53(-/-) cells undergo transformation upon Ras activation. In addition, mouse cells, unlike human cells, express constitutively active telomerase and have long telomeres. However, it is unclear how Ras activation affects human cells of epithelial origin with p53 mutation and/or telomerase activation. In order to address this question, Ha-Ras(G12V) was expressed ectopically in primary as well as hTERT-immortalized human esophageal keratinocytes stably expressing dominant-negative p53 mutants. In human esophageal keratinocytes, we found that Ha-Ras(G12V) induced senescence regardless of p53 status and telomerase activation. Ras activation resulted in changes of cellular morphology, activation of senescence-associated beta-galactosidase, and suppression of cell proliferation, all coupled with reduction in the hyperphosphorylated form of the retinoblastoma protein (pRb). Furthermore, Ha-Ras(G12V) upregulated p16(INK4a) and downregulated cyclin-dependent kinase Cdk4 in human esophageal keratinocytes. Thus, Ras-mediated senescence may involve distinct mechanisms between human and mouse cells. Inactivation of the pRb pathway may be necessary for Ras to overcome senescence and transform human esophageal epithelial cells.

Negative Regulation of Chk2 Expression by p53 Is Dependent on the CCAAT-binding Transcription Factor NF-Y

The kinase Chk2 and tumor suppressor p53 participate in an ill defined regulatory interaction in mammalian cells. The abundance of Chk2 mRNA and protein has now been shown to be decreased by the induction of p53 in Saos2 cells. Ionizing radiation also triggered the phosphorylation and subsequent down-regulation of Chk2 in human colorectal HCT116 (p53(+/+)) cancer cells; irradiation of its isogenic mutant HCT116 (p53(-/-)) cells, which lack functional p53, induced Chk2 phosphorylation but not its down-regulation. In addition, HCT116 (p53(+/+)) cells constitutively expressing a dominant negative p53 (V143A) failed to suppress Chk2 expression after irradiation. Reporter gene assays in HCT116 (p53(+/+)) cells revealed that wild-type p53 repressed, whereas a dominant negative p53 mutant increased, the activity of the human Chk2 gene promoter. Mutational analysis showed that a CCAAT box located between nucleotides -152 and -138 of the promoter was responsible for its negative regulation by p53. Electrophoretic mobility shift assays demonstrated that the transcription factor NF-Y binds to this CCAAT sequence. A dominant negative mutant of NF-YA abolished the effect of p53 on Chk2 promoter activity. These results suggest that p53 negatively regulates Chk2 gene transcription through modulation of NF-Y function and that this regulation may be important for reentry of cells into the cell cycle after DNA damage is repaired.

Genotoxic stress leads to centrosome amplification in breast cancer cell lines that have an inactive G1/S cell cycle checkpoint.

Centrosome amplification plays a key role in the origin of chromosomal instability during cancer development and progression. In this study, breast cancer cell lines with different p53 backgrounds were used to investigate the relationship between genotoxic stress, G(1)/S cell cycle checkpoint integrity, and the development of centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the arrest of both G(1)/S cell cycle progression and centriole duplication. In these cells, which carry functional p53, HU treatment also led to nuclear accumulation of p53 and p21(WAF1), retinoblastoma hypophosphorylation, and downregulation of cyclin A. MCF-7 cells carrying a recombinant dominant-negative p53 mutant (vMCF-7(DNp53)) exhibited a shortened G(1) phase of the cell cycle and retained a normal centrosome phenotype. However, these cells developed amplified centrosomes following HU treatment. The MDA-MB 231 cell line, which carries mutant p53 at both alleles, showed amplified centrosomes at the outset, and developed a hyperamplified centrosome phenotype following HU treatment. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, DR. Taken together, these findings demonstrate that loss of p53 function alone is not sufficient to drive centrosome amplification, but plays a critical role in this process following DNA damage through abrogation of the G(1)/S cell cycle checkpoint. Furthermore, these studies have important clinical implications because they suggest that breast cancers with compromised p53 function may develop centrosome amplification and consequent chromosomal instability following treatment with genotoxic anticancer drugs.

Differential regulation of IGF-II-induced IL-8 by extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases in human keratinocytes

In order to study the relationship between insulin like growth factor-II (IGF-II) and interleukin-8 (IL-8) that are upregulated in psoriasis, we monitored IL-8 expression in IGF-II-treated human keratinocytes and explored the signaling pathways of IL-8 expression by IGF-II. IGF-II increased the IL-8 mRNA and protein levels in human keratinocytes. The upregulation of IL-8 expression by IGF-II was reduced by pretreatment with inhibitors of tyrosine kinase, Src, PI3-kinase, and ERK, but not by p38. Furthermore, IGF-II remarkably increased the DNA binding activities of NF-κB and AP-1, and the IL-8 promoter activity. However, cotransfection with IκB mutant blocked the IGF-II-induced IL-8 promoter activity. In addition, cotransfection with dominant negative MEK1 mutant, but not with dominant negative p38 mutant, blocked the IGF-II-induced IL-8 promoter activity. These results suggest that IGF-II is involved in the pathogenesis of psoriasis by inducing IL-8 gene expression through the tyrosine kinase-Src-ERK1/2-AP-1 pathway, and the PI3-kinase and NF-κB pathway.

P53 deficiency provokes urothelial proliferation and synergizes with activated Ha-ras in promoting urothelial tumorigenesis.

Mutation and deletion of the p53 tumor suppressor gene are arguably the most prevalent among the multiple genetic alterations found in human bladder cancer, but these p53 defects are primarily associated with the advanced diseases, and their roles in bladder tumor initiation and in synergizing with oncogenes in tumor progression have yet to be defined. Using the mouse uroplakin II gene promoter, we have targeted into urothelium of transgenic mice a dominant-negative mutant of p53 that lacks the DNA-binding domain but retains the tetramerization domain. Urothelium-expressed p53 mutant binds to and stabilizes the endogenous wild-type p53, induces nuclear abnormality, hyperplasia and occasionally dysplasia, without eliciting frank carcinomas. Concurrent expression of the p53 mutant with an activated Ha-ras, the latter of which alone induces urothelial hyperplasia, fails to accelerate tumor formation. In contrast, the expression of the activated Ha-ras in the absence of p53, as accomplished by crossing the activated Ha-ras transgenic mice with the p53 knockout mice, results in early-onset bladder tumors that are either low-grade superficial papillary or high grade in nature. These results provide the first in vivo experimental evidence that p53 deficiency predisposes the urothelium to hyperproliferation, but is insufficient for bladder tumorigenesis; that the mere reduction of p53 dosage, as produced in transgenic mice expressing the dominant-negative p53 or in heterozygous p53 knockouts, is incapable of synergizing with Ha-ras to induce bladder tumors; and that the complete loss of p53 is a prerequisite for collaborating with activated Ha-ras to promote bladder tumorigenesis.

Mouse Models Incorporating Alterations In The Major Tumor Suppressor Genes P53 And P16: Their Use In Screening For Potential Carcinogens, Developing Further Relevant Mouse Models, And Screening For Potential Chemopreventive And Chemotherapetutic Agents

This review is primarily a follow up to an initial review on this subject that the authors published 4 years ago [Lubet et al., Exp Lung Res. 2000; 581–593]. The present review gives a brief discussion of certain background points and rationale for development of these specific models, which had been presented in greater detail in the earlier article. In that initial article the authors identified the potential use of mutant mice in screening for carcinogens as well as preventive or therapeutic agents, discussed the relevance of the dominant-negative P53 mutation, as contrasted with knockout P53 mice, and briefly discussed the pros and cons of mice with a germline mutation in tumor suppressor genes in developing mouse models. The primary objective of the present review is to describe more recent studies using mice the dominant-negative P53 mutation as well as to introduce studies with mice with a heterozygous knockout of the P16/Ink4A ARF locus.

Molecular pathways executing the “trophic sentinel” response in HPV-16 E7-expressing normal human diploid fibroblasts upon growth factor deprivation

In response to oncogenic insults, normal human cells execute a defense response that culminates in cellular suicide, apoptosis. Normal human diploid fibroblasts expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein are predisposed to apoptosis when they are deprived of growth factors. Even though a dominant negative p53 mutant abrogates the cell death response, it is not accompanied by p53 phosphorylation, the DNA binding capacity of p53 remains unaltered, and no activation of common p53-dependent transcriptional targets is observed. Expression of two insulin-like growth factor-1 binding proteins, IGFBP-2 and -5, is increased presumably in response to enhanced NF-κB activity in HPV-16 E7-expressing serum-starved cells. Phosphorylation of AKT, an important modulator of IGF-1 survival signaling, is lower in serum-starved E7-expressing cells, and exogenously added IGF-1 can partially inhibit the cell death response. This suggests that IGFBP-2 and -5 may limit IGF-1 availability thus decreasing survival signaling. Caspase 3 but not caspase 8 is activated in serum-starved HPV-16 E7-expressing cells. Caspase inhibition affects nuclear DNA fragmentation, but cell death is not inhibited. Although mitochondria play important roles in caspase-dependent as well as -independent forms of cell death, there is no evidence for cytochrome c release and thus for mitochondrial permeabilization in growth factor deprived HPV-16 E7-expressing cells.

Activation of the stress-activated JNK and p38 MAP kinases in human cells by Photofrin-mediated photodynamic therapy

We have examined the possible role of the stress-activated JNK and p38 protein kinases in cellular sensitivity following Photofrin-mediated photodynamic therapy (PDT). Previously we reported that immortalized Li–Fraumeni syndrome (LFS) cells are more resistant to Photofrin-mediated PDT compared to normal human fibroblasts (NHF) at equivalent cellular Photofrin levels. In the current work we report that Photofrin-mediated PDT increased the activity of JNK1 and p38 within 30 min in both cell types. However, the increased activity of JNK1 and p38 was transient in the sensitive NHF cells and returned back to near basal levels by 3 h after PDT. In contrast, the resistant LFS cells exhibited a more prolonged activation of JNK and p38, which lasted for at least 11 h and 7 h after PDT, respectively. Blocking of the p38 pathway in LFS cells by transient infection with a recombinant adenovirus expressing a dominant negative mutant of p38 or in HeLa cells by stable transfection with a dominant negative mutant of p38 had no effect on cell survival following PDT. These data suggest that although Photofrin-mediated PDT is able to induce JNK1 and p38 in human cells, the p38 pathway alone does not play a major role in the sensitivity of LFS cells to Photofrin-mediated PDT.