HDM2 Protein Research Articles

Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

West Nile virus capsid protein interaction with importin and HDM2 protein is regulated by protein kinase C-mediated phosphorylation

West Nile virus (WNV) capsid (C) protein was shown to enter the nucleus via importin-mediated pathway and induce apoptosis although the precise regulatory mechanisms for such events have remained elusive. In this study, it was shown that WNV C protein was phosphorylated by protein kinase C (PKC). PKC-mediated phosphorylation influenced nuclear trafficking of C protein by modulating the efficiency of C protein–importin-α binding. Combination of bio-informatics, site-directed mutagenesis, co-immunoprecipitation, immuno-fluorescence and mammalian two-hybrid analyses showed that phosphorylation at amino acid residues residing near (Ser83) or within (Ser99 and Thr100) the bipartite nuclear localization motif of WNV C protein was essential for efficient interaction between C protein and importin-α. In addition, phosphorylation of WNV C protein by PKC was shown to enhance its binding to HDM2 and could subsequently induce p53-dependent apoptosis. Collectively, this study highlighted that phosphorylation is an important post-translational modification required to execute the functions of C protein.

Abstract 4534: A novel and potent small molecule antagonist of HDM2, SCH 1450206, activates the p53 pathway with mechanism-based activity

Abstract p53 is an attractive therapeutic target in oncology since aberrant regulation of p53 expression is associated with cancer development and progression. HDM2 is a primary negative regulator of p53 function in cells. The blockade of the p53-HDM2 interaction presents an attractive approach for development of drugs against tumors expressing wild-type p53. Here we report a novel and potent small molecule antagonist of HDM2, SCH 1450206. It binds selectively to the HDM2 protein with high affinity, with weak affinity to HDMX protein. SCH 1450206 induced dose- and time-dependent stabilization of p53 protein and its downstream targets in several human cancer cell lines with wild-type p53, but not in cell with p53 gene deletion. SCH 1450206-induced stabilization of p53 led to distinct changes in expression of p53 target genes regulating apoptosis and cell cycle checkpoints. Inhibition of cell proliferation and induction of apoptosis were surveyed in a panel of human tumor cell lines following SCH 1450206 treatment. Inactivation of p53 by shRNA in cell lines expressing wt-p53 abolished the inhibition of cell proliferation and appearance of senescence in response to SCH 1450206, suggesting the cellular activity of SCH 1450206 is p53 specific and mechanism-based. Taken together, our results demonstrated the mechanism of action of SCH 1450206, a novel HDM2 antagonist that can activate the p53 pathway, and offers a potential anti-tumor agent targeting tumors expressing wild-type p53. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4534.

Abstract 4532: Nucleolin inhibits Hdm2-mediated p53 ubiquitination using multiple interactions

Abstract Nucleolin is a multifunctional abundant nucleolar protein whose transcriptional expression is trans-activated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein has defined roles in ribosomal RNA processing and transcriptional regulation of various genes including rRNA. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its sub-nuclear re-localization. Thus, nucleolin has been demonstrated to have both positive and negative effects on cell proliferation. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2 and inhibits Hdm2-mediated degradation of p53 albeit independent of the ARF (Alternate Reading Frame) tumor suppressor protein. Nucleolin also reduces Hdm2 protein levels by an unknown mechanism when over-expressed in cells to levels found in a variety of cancer cells. Here we have delineated that multiple domains of both nucleolin and Hdm2 are involved in distinct nucleolin-Hdm2 interactions in unstressed cells. Our data indicates that the nucleolin N-terminus as well as the central RNA-binding domain (RBD) are predominantly involved in binding to Hdm2, as determined by Far-Western as well as GST-binding assays in vitro. While the N-terminus of nucleolin was found associated with the Hdm2 RING domain, the nucleolin RBDs robustly bound to the NLS/NES (nuclear localization and export signals) domain of Hdm2. The C-terminal GAR (Glycine-Arginine Rich) domain of nucleolin, shown previously to be essential for association with p53, did not bind Hdm2 yet was found to stimulate Hdm2-mediated p53-ubiquitination. Conversely, the nucleolin RBD that strongly interacted with Hdm2 was found to inhibit the Hdm2 E3 ligase activity against p53, leading to p53 stabilization. There was no significant effect of over-expression of nucleolin or its various domains on Hdm2-autoubiquitination in cells. Similarly, the phosphorylation status of nucleolin did not significantly influence its binding to Hdm2. As might be expected from a key rRNA processing factor, our findings suggest that the activity of nucleolin towards Hdm2 can be modulated to yield either p53 up- or down-regulation. Our studies can aid in the future design of small molecule-based cancer therapeutics that can stimulate the wild-type p53 checkpoint in variety of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4532.

Abstract 1536: Characterization of tumor cell lines selected for resistance to the HDM2 antagonist Nutlin-3a

Abstract The tumor suppressor p53 is activated in response to various cellular stress signals including DNA damage and oncogene activation. Activated p53 regulates the expression of multiple downstream target genes involved in cell cycle control, cell death and senescence. Dysfunction of the p53 pathway is the most frequent alteration in human cancers. HDM2 protein binds directly to p53 and acts as a major negative regulator of the p53 function. Nutlin-3a is an antagonist of the HDM2-p53 protein-protein interaction and restores p53 pathway function in tumors with wild-type p53. Nutlin-3a demonstrates anti-tumor activity both in vitro and in vivo. Emergence of resistance is frequently observed with many anti-cancer therapeutics. The goal of the current studies is to understand the mechanism of resistance to Nutlin-3a. Cancer cell lines were treated with Nutlin-3a for an extended period and resistant clones were isolated. We found using molecular profiling studies that the emergence of Nutlin resistance is primarily due to the inactivation of the p53 signaling pathway through either mutation of the p53 gene or loss of p53 protein expression. These Nutlin-resistant cells are also resistant to others antagonists of the HDM2-p53 protein-protein interaction. These results suggest that, following chronic treatment, resistance to HDM2 antagonists may occur in the clinic. It will be important to combine HDM2 antagonists with other anti-cancer drugs early on during the treatment cycle in order to minimize the emergence of resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1536.

Correlation between pathological data and the RNA expression of p53 or p53-targeted genes in primary invasive ductal breast carcinomas: A preliminary study

The protein expression of the growth suppressive p53 transcription factor and its inhibitor human double minute 2 (Hdm2) is altered in ductal breast carcinomas (DBC). However, the assessment of p53 and/or Hdm2 protein levels in DBC tissues was found to have a questionable prognostic significance. We evaluated the RNA expression of p53, hdm2, and the p53-targeted p21waf-1 and thrombospondin (tsp)-1 by primary DBC tissues, then correlated the RNA levels with patient clinicopathological data. The mean RNA expression level of p53 and that of hdm2 were elevated in large-sized, poorly differentiated, node-positive DBC, while a high p21waf-1 or tsp-1 mean expression level comprised small-sized, low-grade, node-negative tumors. Further analyses found that the correlation between the RNA expression of p53 and that of its targeted genes was reduced as tumor aggressiveness increased. However, for all the examined genes, association of the intensity of RNA expression with the pathological data was not statistically significant (p>0.05). Altogether, our preliminary RNA data confirm the results from previous protein studies, indicating that despite p53 expression and activity show a trend to vary in association with DBC clinical features, neither p53 nor its transcriptional targets can accurately monitor the behaviour of invasive DBC.

TGF-β1–induced expression of human Mdm2 correlates with late-stage metastatic breast cancer

The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%-80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-beta1. Whether TGF-beta1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-beta1-activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-beta1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-beta1-mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-beta1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.

Functional characterization of p53 in nasopharyngeal carcinoma by stable shRNA expression

Nasopharyngeal carcinoma (NPC) is a high-incidence malignancy in Southern China and Southeast Asia. Although mutation of p53 tumor-suppressor gene is a rare event in NPC, NPC has a high frequency of overexpressed/accumulated p53 protein, which was reported to be dysfunction or inactivation in most of NPC. We report here a functional characterization of p53 in an undifferentiated NPC cell line CNE2. To elucidate the biological function of p53, we employed the RNA interference (RNAi) approach to knockdown the endogenously expressed p53 in CNE2 cells. Interestingly, suppression of p53 expression in CNE2 cells was associated with significant down-regulation of p21WAF1/CIP1 expression and decreased HDM2 protein level in both steady state and genotoxic stress induced by ionizing radiation (IR). Consistent with these biochemical data were the accelerated cell cycle progression and the increased proliferation rate, suggesting that p53 retained growth inhibitory activity in CNE2 cells. Indeed, down-regulation of p53 in CNE2 enhanced the ability of CNE2 cells to grow anchorage-independently in vitro and to develop tumors in vivo. Together with the radioresistance acquired by CNE2sip53 cells, our data indicate that in contrast to a previous study, p53 in this NPC cell line remains functional, which may have an important therapeutical implication.

β-Peptides with improved affinity for hDM2 and hDMX

We previously described a series of 3 14-helical β-peptides that bind the hDM2 protein and inhibit its interaction with a p53-derived peptide in vitro. Here we present a detailed characterization of the interaction of these peptides with hDM2 and report two new β-peptides in which non-natural side chains have been substituted into the hDM2-recognition epitope. These peptides feature both improved affinity and inhibitory potency in fluorescence polarization and ELISA assays. Additionally, one of the new β-peptides also binds the hDM2-related protein, hDMX, which has been identified as another key therapeutic target for activation of the p53 pathway in tumors.

Bcl-2 Protects against p53-Induced Apoptosis through Hdm2

Hdm2 is up-regulated in several malignancies including sarcomas and acute myeloid leukemia, where it counteracts the anti-proliferative and pro-apoptotic effect of wild type p53. The anti-apoptotic protein Bcl-2 is often elevated in many tumors with wild type p53 and serves to block p53-induced apoptosis. We demonstrate that the protein level of Hdm2 positively correlates with the level of Bcl-2 and follows the Bcl-2 level in different cell systems. Over-expression of Bcl-2 protects Hdm2 from DNA-damage induced degradation in a dose dependant manner. In addition, modulation of Bcl-2 by shRNA knockdown reduced the Hdm2 protein level in parallel. Consequently, treatment of AML cells with the Bcl-2 small inhibitory molecule HA14-1 attenuated the level of Hdm2. The Bcl-2 level, but not the DNA damage induced Hdm2 degradation, was affected by disruption of the E3 ubiquitin ligase activity of Hdm2. In addition, the DNA-damage induced Hdm2 down-regulation was blocked by disrupted E1 ubiquitin-activation, defect polyubiquitination and by proteasome inhibitors. Finally, we show that Bcl-2 protection from p53-induced cell death requires co-expression of Hdm2 in double null p53/mdm2 mouse embryonic fibroblasts. Our results indicate that Bcl-2 regulates the Hdm2 level and that Hdm2 is a key mediator in Bcl-2 inhibition of p53-induced apoptosis. This is of particular therapeutic interest for cancers displaying elevated Hdm2 and Bcl-2, like sarcoma and acute myeloid leukemia.

Influence of tetramerisation on site-specific post-translational modifications of p53: comparison of human and murine p53 tumour suppressor protein.

The tumour suppressor protein p53 is considered the “Guardian of the Genome”, crucial for cell cycle control and mutated in over 50% of human cancers. Following cellular stress, post-translational modifications such as phosphorylation and acetylation stabilise and activate p53 for cell cycle arrest, DNA repair, apoptosis or senescence. p53 protein functions as a tetramer and we have shown that loss of tetramerisation and changes at the N-terminus influence the recovery of wild type p53 ‘status’. To investigate the relationship between tetramerisation and post-translational modifications we examined a range of site-specific modifications in wild type and dimeric mutant (M340Q/L344R) murine p53 expressed in MEFs p53-/- and in wild type, monomeric (L344P) and dimeric (M340Q/L344R) human p53 expressed in HCT116 p53-/- cells. Using site-specific antibodies we demonstrate that in murine p53, S15 is phosphorylated in a tetramerisation-dependent manner. In contrast, human p53 S15 phosphorylation is not tetramerisation-dependent. Inability to form tetramers in human p53 proteins reduced site-specific N-terminal phosphorylation at S6, S9 and S46 and reduced C-terminal phosphorylation and acetylation at S315 and K382 respectively. In addition, p53 tetramerisation is required for efficient p21 and hdm2 transcription and protein expression and recruitment of p53 to specific promoter regions of p21 and hdm2.

Increased expression of VEGF in a prostate intraepithelial neoplasia (PIN)-like cell line leads to Epithelial-to-Mesenchymal transition (EMT)

The human homologue of murine double minute 2 (HDM2) oncogene is amplified in approximately 7% of all human cancers. Overexpression of HDM2 protein impairs cell cycle control and confers growth advantage to cancer cells. In several cancers the progression of tumor growth and formation of distant metastases are found to be dependent on tumor angiogenesis, a process that is regulated by vascular endothelial growth factor (VEGF). In this study, we have investigated the co-expression of HDM2 and VEGF in various types of human cancer cell lines and have shown that the co-expression is not cell-type-specific. Furthermore, when different types of cell lines were treated with a HDM2 gene specific antisense phosphorothioate oligodeoxynucleotide (HDMAS5), the expression of VEGF mRNA as well as the levels of VEGF protein was found to be decreased. Interestingly, the higher basal levels of VEGF mRNA and the protein observed in HDM2 transfected LNCaP-MST cells were effectively suppressed by HDMAS5 treatment. On the contrary, the mutant oligodeoxynucleotide containing 4 mismatched bases (M4) did not alter the expression of either HDM2 or VEGF in any of the cell lines tested. In conclusion, our findings are the first time evidence showing that HDM2 and VEGF are co-expressed in various cancer cell lines that have aggressive growth and high metastatic abilities. Furthermore, the decrease in VEGF expression observed at the transcriptional as well as translational levels, subsequent to HDMAS5 treatment of p53 null cells, strongly suggests that HDM2 has a regulatory role on VEGF expression in a p53 independent manner.

HDM2 overexpression and promoter polymorphism (SNP309) genotype: Comparison between African American (AA) and Caucasian American (CA) prostate cancer patients (pts)

5133 Background: HDM2 overexpression occurs in a number of tumor types by different mechanisms. The G allele of the HDM2 promoter polymorphism, SNP309 (rs2279744), enhances transcriptional activation, which leads to greater HDM2 expression and accelerated tumor formation at a younger age for patients with Li Fraumeni syndrome and colon cancers. Here we evaluate the HDM2 overexpression in prostate tumors from AA and CA (pts) and examine the association between the G allele with HDM2 protein expression levels, early onset of prostate cancer, and worse clinical outcome. Methods: We studied 51 AA and 50 CA pts prospectively enrolled at the VAMC/New York University. Protein expression of HDM2 in prostate tumor was assessed using immunohistochemistry on tissue microarray. Germline DNA was isolated from normal prostate tissue; a 352bp HDM2 fragment containing SNP309 was amplified; the PCR products were digested with MspA1I restriction enzyme and run on agarose gels. The results were confirmed by DNA sequencing. We calculated HDM2 SNP309 genotype and allele frequencies and examined the association between HDM2 SNP309 genotype, tumor HDM2 protein and clinicopathological features. Results: HDM2 overexpression, defined as HDM2 expression in >10% of tumor cells in 3 cores, was significantly greater in CA pts (78%) vs. AA pts (55%, p=0.0007). The SNP309 allele frequency was not significantly different between AA and CA (81% T, 19% G among the AA pts vs. 72% T, 28% G among CA pts, P=0.41). There was a trend of HDM2 overexpression in AA pt with TG and/or GG compared to TT genotype (63% vs. 37%, respectively, p=0.09) that was not observed in CA pts. No significant associations were observed between a specific genotype and age at diagnosis, tumor stage, Gleason grade, PSA recurrence or death. Conclusions: HDM2 overexpression is significantly more common among CA pts. Although the frequency of the SNP309 G allele is higher in CA pts, in these same pts, HDM2 overexpression does not correlate with presence of the GT or GG genotype. Our data suggest that different mechanisms of HDM2 overexpression might exist in prostate cancer and may operate in pts of different races. No significant financial relationships to disclose.

Reactivation of p53 in cells expressing hepatitis B virus X‐protein involves p53 phosphorylation and a reduction of Hdm2

Multifunctional activities of the hepatitis B virus X-protein (HBx) in cells have been largely implicated in the development of liver cancer; one of these activities is the loss of p53 function by sequestering p53 in the cytoplasm. We have previously found that doxorubicin increased the p53 levels in cells containing p53-binding HBx protein and restored the p53-mediated transcriptional activity that was suppressed by HBx. Here, we investigated the mechanism underlying p53 reactivation. We found that six phosphorylation sites of the Serine residues of p53 were efficiently phosphorylated in HBx-expressing ChangX-34 cells, suggesting that the binding of HBx to the p53 protein does not interfere with the phosphorylation of p53 by signaling kinases. In addition, doxorubicin caused a dramatic reduction of Hdm2 mRNA and protein levels in cells expressing HBx. Intriguingly, reactivation of p53 was accompanied with a nuclear accumulation of p53 and the phosphorylated p53 at Serine15 was only detected in nuclear fraction, but not in cytosolic fraction of doxorubicin-treated ChangX-34 cells. Functional restoration of the p53 protein in HBx-expressing cells occurs according to the dual effects of doxorubicin: a significant reduction of Hdm2 expression and a nuclear accumulation of the phosphorylated p53 protein. Thus, proper usage of doxorubicin as an effective antitumor agent may be reevaluated and can be extended to tumors primarily caused by infection of DNA tumor viruses.

The eIF2α Kinases PERK and PKR Activate Glycogen Synthase Kinase 3 to Promote the Proteasomal Degradation of p53

Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2alpha kinases are critical for mRNA translation, cell proliferation, and apoptosis. Activation of the tumor suppressor p53 results in cell cycle arrest and apoptosis in response to various types of stress. We previously showed that, unlike the majority of stress responses that stabilize and activate p53, induction of endoplasmic reticulum stress leads to p53 degradation through an Mdm2-dependent mechanism. Here, we demonstrate that the endoplasmic reticulum-resident eIF2alpha kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2alpha kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2alpha kinases induce glycogen synthase kinase 3 to promote the nuclear export and proteasomal degradation of p53. Our findings reveal a novel cross-talk between the eIF2alpha kinases and p53 with implications in cell proliferation and tumorigenesis.

Detection of HDM2 and VEGF co-expression in cancer cell lines: novel effect of HDM2 antisense treatment on VEGF expression

The human homologue of murine double minute 2 (HDM2) oncogene is amplified in approximately 7% of all human cancers. Overexpression of HDM2 protein impairs cell cycle control and confers growth advantage to cancer cells. In several cancers the progression of tumor growth and formation of distant metastases are found to be dependent on tumor angiogenesis, a process that is regulated by vascular endothelial growth factor (VEGF). In this study, we have investigated the co-expression of HDM2 and VEGF in various types of human cancer cell lines and have shown that the co-expression is not cell-type-specific. Furthermore, when different types of cell lines were treated with a HDM2 gene specific antisense phosphorothioate oligodeoxynucleotide (HDMAS5), the expression of VEGF mRNA as well as the levels of VEGF protein was found to be decreased. Interestingly, the higher basal levels of VEGF mRNA and the protein observed in HDM2 transfected LNCaP-MST cells were effectively suppressed by HDMAS5 treatment. On the contrary, the mutant oligodeoxynucleotide containing 4 mismatched bases (M4) did not alter the expression of either HDM2 or VEGF in any of the cell lines tested. In conclusion, our findings are the first time evidence showing that HDM2 and VEGF are co-expressed in various cancer cell lines that have aggressive growth and high metastatic abilities. Furthermore, the decrease in VEGF expression observed at the transcriptional as well as translational levels, subsequent to HDMAS5 treatment of p53 null cells, strongly suggests that HDM2 has a regulatory role on VEGF expression in a p53 independent manner.

Myc and Human Papillomavirus Type 16 E7 Genes Cooperate To Immortalize Human Keratinocytes

The E6 protein of the oncogenic human papillomaviruses (HPVs), in combination with the E7 protein, is essential for the efficient immortalization of human foreskin keratinocytes (HFKs). Since we recently demonstrated that E6 activates the human telomerase reverse transcriptase (hTERT) promoter via a Myc-dependent mechanism, we speculated that overexpressed Myc might be able to substitute for E6 in cell immortalization. Myc (similar to E6) was unable to immortalize HFKs when transduced alone, despite inducing high levels of telomerase activity. However, when transduced with E7, Myc immortalized HFKs following a brief but detectable crisis period. In contrast to E6 + E7-immortalized cells, the Myc + E7-immortalized cells expressed high levels of p53 protein as well as two p53-regulated proteins, p21 and hdm-2. The increase in p21 and hdm-2 proteins correlated directly with their mRNA levels, suggesting transcriptional activation of the respective genes by the overexpressed p53 protein. Interestingly, a significant proportion of the p53 protein in the Myc + E7-immortalized cells was localized to the cytoplasm, potentially due to interactions with the overexpressed hdm-2 protein. Regardless, cell immortalization by the Myc + E7 genes occurred independently of p53 degradation. Since we have already observed high-efficiency cell immortalization with the hTERT + E7 or E6 mutant (p53 degradation-defective) + E7 genes (i.e., no crisis period) that proceeds in the presence of high levels of p53, we hypothesize that the crisis period in the Myc + E7 cells is due not to the levels of the p53 protein but rather to unique properties of the Myc protein. The common factor in cell immortalization by the three gene sets (E6 + E7, Myc + E7, and hTERT + E7 genes) is the induction of telomerase activity.

Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML.Hdm2 and pro-apoptotic Bcl-2 members are among the most intensely induced genes immediately after chemotherapy and Hdm2 is proposed a role in receptor tyrosine kinase regulation. Thus we examined the DNA damage related modulation of these proteins in relation to FLT3 mutational status and induction of apoptosis.ResultsWithin one hour after exposure to ionizing radiation (IR), the AML cells (NB4, MV4-11, HL-60, primary AML cells) showed an increase in Flt3 protein independent of mRNA levels, while the Hdm2 protein decreased. The FLT3 mutant MV4-11 cells were resistant to IR accompanied by presence of both Mcl-1 and Hdm2 protein three hours after IR. In contrast, the FLT3 wild type NB4 cells responded to IR with apoptosis and pre-apoptotic Mcl-1 down regulation. Daunorubicin (DNR) induced continuing down regulation of Hdm2 and Mcl-1 in both cell lines followed by apoptosis.ConclusionBoth IR and DNR treatment resulted in concerted protein modulations of Mcl-1, Hdm2 and Flt3. Cell death induction was associated with persistent attenuation of Mcl-1 and Hdm2. These observations suggest that defining the pathway(s) modulating Flt3, Hdm2 and Mcl-1 may propose new strategies to optimize therapy for the relapse prone FLT3 mutated AML patients.

Roscovitine‐activated HIP2 kinase induces phosphorylation of wt p53 at Ser‐46 in human MCF‐7 breast cancer cells

Human MCF-7 breast cancer cells are relatively resistant to conventional chemotherapy due to the lack of caspase-3 activity. We reported recently that roscovitine (ROSC), a potent cyclin-dependent kinase 2 inhibitor, arrests human MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis. Exposure of MCF-7 cells to ROSC also strongly activates the wt p53 tumor suppressor protein in a time- and dose-dependent manner. The p53 level increased despite upregulation of Hdm-2 protein and was attributable to the site-specific phosphorylation at Ser-46. The p53 protein phosphorylated at serine 46 causes the up-regulation of the p53AIP1 protein, a component of mitochondria. In the present study we identified the pathway mediating ROSC-induced p53 activation. Exposure of MCF-7 cells to ROSC activated homeodomain-intereacting protein kinase-2 (HIPK2). The overexpression of wild-type but not kinase inactive HIPK2 increased the basal and ROSC-induced level of p53 phosphorylation at Ser-46 and strongly enhanced the rate of apoptosis in cells exposed to ROSC. We show that HIPK2 is activated by ROSC and mediates ROSC-induced P-Ser-46-p53, thereby stabilizing wt p53 and increasing the efficacy of drug-induced apoptosis in MCF-7 cells. These results identify HIPK2 as a component of the ROSC-induced signaling pathway leading to the stabilization and activation of wt p53 protein.

Protoporphyrin IX Interacts with Wild-type p53 Protein in Vitro and Induces Cell Death of Human Colon Cancer Cells in a p53-dependent and -independent Manner

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.