Sylvia Wilder

p53-mediated cell death: relationship to cell cycle control.

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.

A novel p53‐inducible gene, PAG608, encodes a nuclear zinc finger protein whose overexpression promotes apoptosis

The biological effects of the p53 tumor suppressor protein are elicited, at least in part, through sequence‐specific transactivation of a battery of target genes. The differential display method was employed towards identifying additional p53 target genes, with emphasis on genes whose induction may contribute to p53‐mediated apoptosis. We report here the cloning of a novel p53‐inducible gene, designated PAG608. PAG608 transcripts are induced by DNA damage in a p53‐dependent manner. PAG608 encodes a nuclear zinc finger protein, which appears to localize preferentially to nucleoli when expressed at moderate levels in transfected cells. Transient overexpression of PAG608 in human tumor‐derived cells leads to distinctive changes in nuclear morphology, and can promote apoptosis. Together with additional p53 target genes, PAG608 may therefore play a role in mediating the biological activities of p53.

Mutant p53 Prolongs NF-κB Activation and Promotes Chronic Inflammation and Inflammation-Associated Colorectal Cancer

The tumor suppressor p53 is frequently mutated in human cancer. Common mutant p53 (mutp53) isoforms can actively promote cancer through gain-of-function (GOF) mechanisms. We report that mutp53 prolongs TNF-α-induced NF-κB activation in cultured cells and intestinal organoid cultures. Remarkably, when exposed to dextran sulfate sodium, mice harboring a germline p53 mutation develop severe chronic inflammation and persistent tissue damage, and are highly prone to inflammation-associated colon cancer. This mutp53 GOF is manifested by rapid onset of flat dysplastic lesions that progress to invasive carcinoma with mutp53 accumulation and augmented NF-κB activation, faithfully recapitulating features frequently observed in human colitis-associated colorectal cancer (CAC). These findings might explain the early appearance of p53 mutations in human CAC.

p53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation

The p53 tumor suppressor is mutated in a high percentage of human tumors. However, many other tumors retain wild-type (wt) p53 expression, raising the intriguing possibility that they actually benefit from it. Recent studies imply a role for p53 in regulation of autophagy, a catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles, particularly under conditions of nutrient deprivation. Here, we show that, in many cell types, p53 confers increased survival in the face of chronic starvation. We implicate regulation of autophagy in this effect. In HCT116 human colorectal cancer cells exposed to prolonged nutrient deprivation, the endogenous wt p53 posttranscriptionally down-regulates LC3, a pivotal component of the autophagic machinery. This enables reduced, yet sustainable autophagic flux. Loss of p53 impairs autophagic flux and causes excessive LC3 accumulation upon starvation, culminating in apoptosis. Thus, p53 increases cell fitness by maintaining better autophagic homeostasis, adjusting the rate of autophagy to changing circumstances. We propose that some cancer cells retain wt p53 to benefit from the resultant increased fitness under limited nutrient supply.

RNF20 and USP44 Regulate Stem Cell Differentiation by Modulating H2B Monoubiquitylation

Embryonic stem cells (ESCs) maintain high genomic plasticity, which is essential for their capacity to enter diverse differentiation pathways. Posttranscriptional modifications of chromatin histones play a pivotal role in maintaining this plasticity. We now report that one such modification, monoubiquitylation of histone H2B on lysine 120 (H2Bub1), catalyzed by the E3 ligase RNF20, increases during ESC differentiation and is required for efficient execution of this process. This increase is particularly important for the transcriptional induction of relatively long genes during ESC differentiation. Furthermore, we identify the deubiquitinase USP44 as a negative regulator of H2B ubiquitylation, whose downregulation during ESC differentiation contributes to the increase in H2Bub1. Our findings suggest that optimal ESC differentiation requires dynamic changes in H2B ubiquitylation patterns, which must occur in a timely and well-coordinated manner.

Prevalent and rare mutations among Gaucher patients

Sequence analysis of gcs cDNA (encoding glucocerebrosidase) or genomic fragments originated from Gaucher patients revealed novel mutations. Two rare mutations were found in a type-2 non-Jewish Gaucher patient: a G----A transition (Gly325----Arg) at nucleotide (nt) 5306 of the active gene and a T----G transversion (Cys342----Gly) at nt 5357. These mutations were not found in any other patient. A G----C transversion (Asp409----His) at nt 5957 was identified in two non-Jewish patients, and was designated TL. Two recombinant alleles were found. One recombinant allele designated recTL contained four single-nt mutations. These mutations included: (1) a G----C transversion at nt 5957 (Asp409----His) (the TL mutation); (2) a T----C transition at nt 6433 (Leu444----Pro) creating a new NciI site (NciI mutation); (3) a G----C transversion at nt 6468 (Ala456----Pro; 456 mutation); and (4) a G----C transversion at amino acid (aa) 460 (nt 6482), not associated with any aa change. Sequence analysis indicated that at least part of exon 9, intron 9 and exon 10 of the recombinant gene derived from the pseudogene. The other recombinant gene, designated recNciI, contained a mutation at aa 444 (NciI mutation), and mutations 456 and 460 described above; at least exon 10 of this gene originated from the pseudogene. We hypothesize that the presence of the pseudogene close to the active gene causes transfer of mutations into the active gene via gene conversion or nonhomologous recombination, thus accounting for the high frequency of mutations observed in the gcs gene.

The c-fos Proto-Oncogene Is a Target for Transactivation by the p53 Tumor Suppressor

ABSTRACT The p53 tumor suppressor gene is mutated in over 50% of human cancers, resulting in inactivation of the wild-type (wt) p53 protein. The most notable biochemical feature of p53 is its ability to act as a sequence-specific transcriptional activator. Through use of the suppression subtractive hybridization differential screening technique, we identified c-fos as a target for transcriptional stimulation by p53 in cells undergoing p53-mediated apoptosis. Overexpression of wt p53 induces c-fos mRNA and protein. Moreover, in vivo induction of c-fos in the thymus following whole-body exposure to ionizing radiation is p53 dependent. p53 responsiveness does not reside in the basal c-fos promoter. Rather, a distinct region within the c-fos gene first intron binds specifically to p53 and confers upon the c-fos promoter the ability to become transcriptionally activated by wt p53. Identification of c-fos as a specific target for transcriptional activation by p53 establishes a direct link between these two pivotal regulatory proteins and raises the possibility that c-fos contributes to some of the biological effects of p53.

Importin 7 and Exportin 1 Link c-Myc and p53 to Regulation of Ribosomal Biogenesis

Members of the β-karyopherin family mediate nuclear import of ribosomal proteins and export of ribosomal subunits, both required for ribosome biogenesis. We report that transcription of the β-karyopherin genes importin 7 (IPO7) and exportin 1 (XPO1), and several additional nuclear import receptors, is regulated positively by c-Myc and negatively by p53. Partial IPO7 depletion triggers p53 activation and p53-dependent growth arrest. Activation of p53 by IPO7 knockdown has distinct features of ribosomal biogenesis stress, with increased binding of Mdm2 to ribosomal proteins L5 and L11 (RPL5 and RPL11). Furthermore, p53 activation is dependent on RPL5 and RPL11. Of note, IPO7 and XPO1 are frequently overexpressed in cancer. Altogether, we propose that c-Myc and p53 counter each other in the regulation of elements within the nuclear transport machinery, thereby exerting opposing effects on the rate of ribosome biogenesis. Perturbation of this balance may play a significant role in promoting cancer.

Three unique base pair changes in a family with Gaucher disease.

SummarySingle-stranded cDNA was prepared from RNA obtained from a patient with type 1 Gaucher disease. The cDNA was amplified in vitro and analyzed by sequencing. Three base-pair changes were identified which included a G to C transversion at nucleotide 3119 of the active gene (Asp140→His), an A to C transversion at nucleotide 3170 (Lys157→Gln) and a G to A change at nucleotide 5309 (Glu326→Lys). To study the mode of inheritance of the three different base-pair changes, genomic DNA was prepared from blood or skin fibroblasts of several family members. Genomic glucocerebrosidase DNA sequences were amplified and subjected to hybridization with allele-specific oligonucleotides (ASOs). The hybridization profiles demonstrated that two of the basepair changes originated from the mother and were transmitted to her two affected sons and to a grandchild, while the third base-pair change, originating from the father, was transmitted to his two affected sons, a carrier daughter and a second grandchild. Tests of other patients with Gaucher disease failed to disclose the presence of the three base-changes. This is a unique family with three base-pair changes tightly linked to Gaucher disease.

The PI3K inhibitor LY294002 prevents p53 induction by DNA damage and attenuates chemotherapy-induced apoptosis

The p53 tumor suppressor plays a key role in the natural protection against cancer. Activation of p53 by DNA-damaging agents can contribute to successful elimination of cancer cells via chemotherapy-induced apoptosis. The phosphatidylinositol-3 kinase (PI3K) pathway, triggered in normal cells upon exposure to growth factors, regulates a cascade of proliferation and survival signals. The PI3K pathway is abnormally active in many cancers, thus making it an attractive target for inactivation in an attempt to achieve better cancer therapy. We report here that exposure to LY294002, a potent PI3K inhibitor, aborts the activation of p53 by several drugs commonly used in cancer chemotherapy. Concomitantly, LY294002 attenuates p53-dependent, chemotherapy-induced apoptosis of cancer cells. These findings invoke an unexpected positive role for PI3K in p53 activation by anticancer agents, and suggest that the efficacy of PI3K inhibitors in cancer therapy may be greatly affected by the tumor p53 status.