A role for miR-33 in p53 regulation: New perspectives for hematopoietic stem cell research

Comment on: Herrera-Merchan A, et al. Cell Cycle 2010; 9:3277-85.     

A small molecule inhibitor of p53-inducible protein phosphatase PPM1D

PPM1D is a p53-inducible Ser/Thr protein phosphatase. PPM1D gene amplification and overexpression have been reported in a variety of human tumors, including breast cancer and neuroblastoma. Because the phosphatase activity of PPM1D is essential for its oncogenic role, PPM1D inhibitors should be viable anti-cancer agents. In our current study, we showed that SPI-001 was a potent and specific PPM1D inhibitor. SPI-001 inhibited PPM1D phosphatase activity in PPM1D-overexpressing human breast cancer cells and increased phosphorylation of p53. Furthermore, SPI-001 suppressed cell proliferation by inducing apoptosis. Our present study suggested that SPI-001 was a potential lead compound in developing anti-cancer drugs.     

Akt: A double-edged sword for hematopoietic stem cells

The phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway is dysregulated in a wide range of malignancies, including leukemia, and several members of this pathway are attractive therapeutic targets in oncology1. Although Akt is constitutively phosphorylated in up to 90% of cases of acute myeloid leukemia (AML), its role in AML has been unclear2. In some cases, Akt activation is attributed to activation of tyrosine kinases, such as BCR-ABL or FLT3-ITD, but in many cases the mechanism of Akt phosphorylation and its significance is unknown. Is AKT activation simply a marker of leukemic transformation, or is it a driver of leukemogenesis?     

The ups and downs of p53 regulation in hematopoietic stem cells

Hematopoietic stem cells provide an indispensible source for replenishing the blood with all its constituents throughout the organism's lifetime. Mice with a compromised hematopoietic stem cell compartment cannot survive. p53, a major tumor suppressor gene, has been implicated in regulation of hematopoiesis. In particular, p53 plays a role in homeostasis by regulating HSC quiescence and self renewal. We recently utilized a hypomorphic p53^515C allele in conjunction with Mdm2, a negative regulator of p53 to gain insights into the role of p53 in hematopoietic regulation. Our analyses revealed that p53^515C/515CMdm2^-/- double mutant mice die soon after birth due to hematopoietic failure. Further mechanistic studies revealed that in the absence of Mdm2, ROS induced postnatal p53 activity depletes hematopoietic stem cells, progenitors and differentiated cells.     

The ups and downs of p53 regulation in hematopoietic stem cells

Hematopoietic stem cells provide an indispensible source for replenishing the blood with all its constituents throughout the organism''s lifetime. Mice with a compromised hematopoietic stem cell compartment cannot survive. p53, a major tumor suppressor gene, has been implicated in regulation of hematopoiesis. In particular, p53 plays a role in homeostasis by regulating HSC quiescence and self renewal. We recently utilized a hypomorphic p53^515C allele in conjunction with Mdm2, a negative regulator of p53, to gain insights into the role of p53 in hematopoietic regulation. Our analyses revealed that p53^515C/515CMdm2^−/− double mutant mice die soon after birth due to hematopoietic failure. Further mechanistic studies revealed that in the absence of Mdm2, ROS-induced postnatal p53 activity depletes hematopoietic stem cells, progenitors and differentiated cells.Key words: HSC, reactive oxygen species, ROS, p53, Mdm2     

Adenosine triphosphate stimulates thymidine incorporation but does not promote cell growth in primary cultures of renal proximal tubule cells.

Acute addition of adenosine triphosphate (ATP) stimulated thymidine incorporation in confluent, quiescent primary cultures of rabbit renal proximal tubule cells in a dose-responsive manner. Similar increases in thymidine incorporation was observed with adenosine diphosphate and adenosine monophosphate but not with adenosine. The effect of chronic administration of ATP, however, suppressed cell growth. This suppression appears to be due to an effect of ATP to cause detachment of cells from culture plates, resulting in an increase in thymidine incorporation acutely but in suppression of cell growth chronically. ATP is, therefore, not a direct growth promoter of renal proximal tubule cells in primary culture.     

ADP-ribosylation of p53 tumor suppressor protein: Mutant but not wild-type p53 is modified

Poly(ADP-ribosyl)ation of mutant and wild-type p53 was studied in transformed and nontransformed rat cell lines constitutively expressing the temperature-sensitive p53^135val. It was found that in both cell types at 37.5°C, where overexpressed p53 exhibits mutant conformation and cytoplasmic localization, a considerable part of the protein was poly(ADP-ribosyl)ated. Using densitometric scanning, the molecular mass of the modified protein was estimated as 64 kD. Immunofluorescence studies with affinity purified anti-poly(ADP-ribose) transferase (pADPRT) antibodies revealed that, contrary to predictions, the active enzyme was located in the cytoplasm, while in nuclei chromatin was depleted of pADPRT. A distinct intracellular localization and action of pADPRT was found in the cell lines cultivated at 37.5°C, where p53 adopts wild-type form. Despite nuclear coexistence of both proteins no significant modification of p53 was found. Since the strikingly shared compartmentalization of p53 and pADPRT was indicative of possible complex formation between the two proteins, reciprocal immunoprecipitation and immunoblotting were performed with anti-p53 and anti-pADPRT antibodies. A poly(ADP-ribosyl)ated protein of 116 kD constantly precipitated at stringent conditions was identified as the automodified enzyme. It is concluded that mutant cytoplasmic p53 is tighly complexed to pADPRT and becomes modified. At 32.5°C binding to DNA of p53 or its temperature-dependent conformational alteration might prevent an analogous modification of the tumor suppressor protein. © 1996 Wiley-Liss, Inc.     

Role for Geminin in sustaining the activity of hematopoietic stem cells

Ohno Y, et al. Proc Natl Acad Sci USA 2010; In press.     

Identification of a selective small molecule inhibitor of breast cancer stem cells

A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).     

CP-31398 restores DNA-binding activity to mutant p53 in vitro but does not affect p53 homologs p63 and p73

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effective in vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elements in vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (B(max)) and its affinity (K(d)) for DNA. The compound, however, does not affect the affinity (K(d) value) of wild type p53 for DNA and only increases B(max) slightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.     

p53: Guardian of reprogramming

The reprogramming of somatic cells to induced pluripotent stem (iPS) cells is one of the major discoveries of recent years. The development and application of patient specific iPS lines could potentially revolutionize cell-based therapy, facilitating the treatment of a wide range of diseases. Despite the numerous technological advancements in the field, an in-depth mechanistical understanding of the pathways involved in reprogramming is still lacking. Several groups have recently provided a mechanistical insight into the role of the p53 tumor suppressor pathway in reprogramming. The repercussions of these findings are profound and reveal an unexpected role of p53 as a "guardian of reprogramming", ensuring genomic integrity during reprogramming at the cost of a reduced efficiency of the process. Here we analyze the latest findings in the field and discuss their relevance for future applications of iPS cell technology.     

A putative protein inhibitor of activated STAT (PIASy) interacts with p53 and inhibits p53-mediated transactivation but not apoptosis

The p53 protein has recently been reported to be capable of mediating apoptosis through a pathway that is not dependent on its transactivation function. We report here that the PIASy member of the protein inhibitor of activated STAT family inhibited p53's transactivation function without compromising its ability to induce apoptosis of the H1299 nonsmall cell lung carcinoma cell line. The p53 protein bound to PIASy in yeast two-hybrid assays and coprecipitated in complexes with p53 in immunoprecipitates from mammalian cells. PIASy inhibited the DNA-binding activity of p53 in nuclear extracts and blocked the ability of p53 to induce expression of two of its target genes, Bax and p21^Waf1/Cip1, in H1299 cells. The block in p53-mediated induction of Bax and p21 was determined to be at the level of transactivation, since PIASy inhibited p53's ability to transactivate a p21/luciferase reporter construct. PIASy did not effect the incidence of apoptosis in H1299 cells upregulated for p53. PIASy appears to regulate p53-mediated functions and may direct p53 into a transactivation-independent mode of apoptosis.     

Genotoxic aldehyde stress prematurely ages hematopoietic stem cells in a p53-driven manner

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The p53 tumor suppressor protein is a critical regulator of hematopoietic stem cell behavior

In response to diverse stresses, the tumor suppressor p53 differentially regulates its target genes, variably inducing cell-cycle arrest, apoptosis or senescence. Emerging evidence indicates that p53 plays an important role in regulating hematopoietic stem cell (HSC) quiescence, self-renewal, apoptosis and aging. The p53 pathway is activated by DNA damage, defects in ribosome biogenesis, oxidative stress and oncogene induced p19ARF upregulation. We present an overview of the current state of knowledge about p53 (and its target genes) in regulating HSC behavior, with the hope that understanding the molecular mechanisms that control p53 activity in HSCs and how p53 mutations affect its role in these events may facilitate the development of therapeutic strategies for eliminating leukemia (and cancer) propagating cells.