Behavioral alterations associated with apoptosis and down-regulation of presenilin 1 in the brains of p53-deficient mice

Presenilin 1 (PS1) expression is repressed by the p53 tumor suppressor. As shown herein, wild-type PS1 is an effective antiapoptotic molecule capable of significantly inhibiting p53-dependent and p53-independent cell death. We analyzed, at the functional and molecular levels, the brains of p53 knockout mice. Surprisingly, we found that lack of p53 expression induces apoptotic brain lesions, accompanied by learning deficiency and behavioral alterations. p53-deficient mice show an unexpected overexpression of p21waf1 with subsequent down-regulation of PS1 in their brains. This process is progressive and age-dependent. These data indicate that the p53 pathway, besides affecting tumor suppression, may play a major role in regulating neurobehavioral function and cell survival in the brain.

c-Abl is required for development and optimal cell proliferation in the context of p53 deficiency

The c-Abl tyrosine kinase and the p53 tumor suppressor protein interact functionally and biochemically in cellular genotoxic stress response pathways and are implicated as downstream mediators of ATM (ataxia-telangiectasia mutated). This fact led us to study genetic interactions in vivo between c-Abl and p53 by examining the phenotype of mice and cells deficient in both proteins. c-Abl-null mice show high neonatal mortality and decreased B lymphocytes, whereas p53-null mice are prone to tumor development. Surprisingly, mice doubly deficient in both c-Abl and p53 are not viable, suggesting that c-Abl and p53 together contribute to an essential function required for normal development. Fibroblasts lacking both c-Abl and p53 were similar to fibroblasts deficient in p53 alone, showing loss of the G1/S cell-cycle checkpoint and similar clonogenic survival after ionizing radiation. Fibroblasts deficient in both c-Abl and p53 show reduced growth in culture, as manifested by reduction in the rate of proliferation, saturation density, and colony formation, compared with fibroblasts lacking p53 alone. This defect could be restored by reconstitution of c-Abl expression. Taken together, these results indicate that the ATM phenotype cannot be explained solely by loss of c-Abl and p53 and that c-Abl contributes to enhanced proliferation of p53-deficient cells. Inhibition of c-Abl function may be a therapeutic strategy to target p53-deficient cells selectively.

Identification of a novel p53 functional domain that is necessary for efficient growth suppression

Activation of the p53 tumor suppressor protein has been demonstrated to block cell growth by inducing either a transient cell cycle arrest or programmed cell death (apoptosis). Although evidence exists linking p53’s function as an activator of transcription to its ability to effect cell cycle arrest, the role of this activity in the induction of apoptosis remains unclear. To gain insight into the molecular mechanisms underlying p53-mediated antiproliferative pathways, a study was initiated to explore the functions of a putative p53 signaling domain. This region of the human p53 protein is localized between amino acids 61 and 94 (out of 393) and is noteworthy in that it contains five repeats of the sequence PXXP (where P represents proline and X any amino acid). This motif has been shown to play a role in signal transduction via its SH3 domain binding activity. A p53 cDNA deletion mutant (ΔproAE), which lacks this entire proline-rich domain (deleted for amino acids 62–91), was created and characterized for a variety of p53 functions. The entire domain has been shown to be completely dispensable for transcriptional activation. On the other hand, this deletion of the p53 proline-rich domain impairs p53’s ability to suppress tumor cell growth in culture. Amino acid substitution mutations at residues 22 and 23 of p53 (eliminates transcriptional activity) also impair p53-mediated inhibition of cell growth in culture. Unlike wild-type p53, the ΔproAE mutant cDNA can be stably expressed in tumor derived cell lines with few immediate detrimental effects. These cells express physiologic levels of p53 protein that are induced normally in response to DNA damage, indicating that removal of the proline-rich domain does not disrupt p53’s upstream regulation by DNA damage. These data indicate that, in addition to the transcriptional activation domain, the p53 proline-rich domain plays a critical role in the transmission of antiproliferative signals downstream of the p53 protein and may link p53 to a direct signal transduction pathway.

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32–36°, whereas wt p53 bends it by 51–57°. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by ≈35° and ≈70°, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53–DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.

p53 regulates a G2 checkpoint through cyclin B1

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, we have derived a human ovarian cell that undergoes p53-dependent G2 arrest at 32°C. We have found that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation.

Activation of p53 in cervical carcinoma cells by small molecules

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21WAF1/CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.

Opposing roles of pRB and p107 in adipocyte differentiation

The retinoblastoma (RB) family of proteins, pRB, p107, and p130, have been postulated to be partially redundant in their ability to regulate progression through the G1 phase of the cell cycle. However, pRB appears to be unique in its capacity as a classical tumor suppressor, possibly because of a specialized role in maintaining the balance between proliferation and differentiation. A variety of studies have in fact revealed an apparent role for pRB in cellular differentiation and development. However, roles for p107 and p130 in differentiation have not yet been established, and knockout mouse studies have indicated that they may be functionally redundant during development, and possibly perform a role in differentiation distinct from that of pRB. Using adipogenesis as a model, we have indeed found distinct roles for the pRB family proteins in regulating differentiation. 3T3 fibroblasts deficient in p107 and p130 differentiate with high efficiency, whereas pRB−/− 3T3 cells exhibit defects in their differentiation potential. Moreover, over-expression of pRB in wild-type cells promotes differentiation, whereas over-expression of p107 antagonizes differentiation. The seemingly opposing roles of pRB family members in adipocyte differentiation can be explained, at least in part, by a requirement for pRB in maintaining cell cycle exit as well as potentiating the activity of the differentiation-associated transcription factor, C/EBPα. p107 does not affect C/EBPα-driven transcription and is not required for cell cycle exit, but instead, loss of p107 lowers the requirement for the differentiation factor PPARγ. These findings suggest contrasting biological roles for individual members of the pRB family of proteins that may explain why pRB, but not p107, is commonly mutated during human tumor development.

Identification of an additional negative regulatory region for p53 sequence-specific DNA binding

The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80–93 and 364–393. The C-terminal residues 364–393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80–93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80–93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80–93 and 364–393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.

MEKK1/JNK signaling stabilizes and activates p53

Activation of the tumor suppressor p53 by stress and damage stimuli often correlates with induction of stress kinases, Jun-NH2 kinase (JNK). As JNK association with p53 plays an important role in p53 stability, in the present study we have elucidated the relationship between the JNK-signaling pathway and p53 stability and activity. Expression of a constitutively active form of JNKK upstream kinase, mitogen-activated protein kinase kinase kinase (ΔMEKK1), increased the level of the exogenously transfected form of p53 in p53 null (10.1) cells as well as of endogenous p53 in MCF7 breast cancer cells. Increased p53 level by forced expression of ΔMEKK1 coincided with a decrease in p53 ubiquitination in vivo and with prolonged p53 half-life. Computerized modeling of the JNK-binding site (amino acids 97–116; p7 region) enabled us to design mutations of exposed residues within this region. Respective mutations (p53101-5-8) and deletion (p53Δp7) forms of p53 did not exhibit the same increase in p53 levels upon ΔMEKK1 expression. In vitro phosphorylation of p53 by JNK abolished Mdm2 binding and targeting of p53 ubiquitination. Similarly, ΔMEKK1 expression increased p53 phosphorylation by immunopurified JNK and dissociated p53–Mdm2 complexes. Transcriptional activity of p53, as measured via mdm2 promoter-driven luciferase, exhibited a substantial increase in ΔMEKK1-expressing cells. Cotransfection of p53 and ΔMEKK1 into p53 null cells potentiated p53-dependent apoptosis, suggesting that MEKK1 effectors contribute to the ability of p53 to mediate programmed cell death. Our results point to the role of MEKK1-JNK signaling in p53 stability, transcriptional activities, and apoptotic capacity as part of the cellular response to stress.

The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules. Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium.

Adenovirus E4orf6 oncoprotein modulates the function of the p53-related protein, p73

Recently, several proteins have been identified that are related in their sequence to the p53 tumor-suppressor protein. One of these proteins, which is termed p73, exhibits sequence homology to the p53 transcriptional activation, DNA binding, and oligomerization domains. The adenovirus E1B 55-kDa protein, the adenovirus E4orf6 protein, and SV40 T antigen each can bind to p53 and inhibit p53 function. Here we demonstrate that the adenovirus E4orf6 protein, but not the E1B 55-kDa protein or T antigen, interacts with p73. The E4orf6 protein inhibits p73-mediated transcriptional activation and cell killing in a manner similar to its effect on p53. Thus, only a subset of viral oncoproteins that antagonize p53 function also interacts with the related p73 protein.

p53 Accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101

Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101−/− embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101−/− embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21WAF-1/CIP-1, was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101−/− embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.

Genetic disruption of PPARδ decreases the tumorigenicity of human colon cancer cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that have been implicated in a variety of biologic processes. The PPARδ isotype was recently proposed as a downstream target of the adenomatous polyposis coli (APC)/β-catenin pathway in colorectal carcinogenesis. To evaluate its role in tumorigenesis, a PPARδ null cell line was created by targeted homologous recombination. When inoculated as xenografts in nude mice, PPARδ −/− cells exhibited a decreased ability to form tumors compared with PPARδ +/− and wild-type controls. These data suggest that suppression of PPARδ expression contributes to the growth-inhibitory effects of the APC tumor suppressor.

The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation

The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.

p19ARF targets certain E2F species for degradation

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.