Eitan Shaulian

AP-1 in cell proliferation and survival

A plethora of physiological and pathological stimuli induce and activate a group of DNA binding proteins that form AP-1 dimers. These proteins include the Jun, Fos and ATF subgroups of transcription factors. Recent studies using cells and mice deficient in individual AP-1 proteins have begun to shed light on their physiological functions in the control of cell proliferation, neoplastic transformation and apoptosis. Above all such studies have identified some of the target genes that mediate the effects of AP-1 proteins on cell proliferation and death. There is evidence that AP-1 proteins, mostly those that belong to the Jun group, control cell life and death through their ability to regulate the expression and function of cell cycle regulators such as Cyclin D1, p53, p21cip1/waf1, p19ARF and p16. Amongst the Jun proteins, c-Jun is unique in its ability to positively regulate cell proliferation through the repression of tumor suppressor gene expression and function, and induction of cyclin D1 transcription. These actions are antagonized by JunB, which upregulates tumor suppressor genes and represses cyclin D1. An especially important target for AP-1 effects on cell life and death is the tumor suppressor p53, whose expression as well as transcriptional activity, are modulated by AP-1 proteins.

The Mammalian UV Response: c-Jun Induction Is Required for Exit from p53-Imposed Growth Arrest

The mammalian UV response results in rapid and dramatic induction of c-jun. Induction of a protooncogene, normally involved in mitogenic responses, by a genotoxic agent that causes growth arrest seems paradoxical. We now provide an explanation for the role of c-Jun in the UV response of mouse fibroblasts. c-Jun is necessary for cell-cycle reentry of UV-irradiated cells, but does not participate in the response to ionizing radiation. Cells lacking c-Jun undergo prolonged cell-cycle arrest, but resist apoptosis, whereas cells that express c-Jun constitutively do not arrest and undergo apoptosis. This function of c-Jun is exerted through negative regulation of p53 association with the p21 promoter. Cells lacking c-Jun exhibit prolonged p21 induction, whereas constitutive c-Jun inhibits UV-mediated p21 induction.

Induction of Mdm2 and enhancement of cell survival by bFGF

Basic fibroblast growth factor (bFGF) can exert mitogenic and viability-promoting effects in a wide range of biological systems. The biochemical activities mediating the cell survival function of bFGF are largely unknown. We report here that exposure of fibroblasts to bFGF, which confers upon them increased survival, also causes at the same time an increase in cellular levels of the Mdm2 oncoprotein. Cells constitutively exposed to a bFGF autocrine loop are more refractory to killing by cisplatin. This increased chemoresistance coincides with elevated Mdm2 and reduced activation of the endogenous p53, resulting in inefficient transcriptional activation of the bax gene promoter. Importantly, unlike Mdm2 accumulation in fibroblasts exposed to DNA damage, induction of Mdm2 by bFGF does not occur through a p53-mediated pathway. The role of p53 in DNA damage-induced apoptosis and the ability of Mdm2 to block p53-mediated cell death are well established. These findings therefore suggest that induction of Mdm2 and the subsequent inhibition of p53 function may contribute, at least partially, to the anti-apoptotic effects of bFGF and possibly some other survival factors.

Stress-induced JNK Activation Is Independent of Gadd45 Induction

DNA damage and environmental stress activate signaling and induce genes involved in cell cycle and cell death. Expression of the Gadd45 protein is induced following DNA damage and other stress. Gadd45 is believed to play a role in growth arrest and possibly in cell death. The JNK signaling pathway is also activated by some DNA-damaging agents. This activation leads to phosphorylation and activation of transcription factors, such as c-Jun/AP-1 and ATF2, which mediate immediate early gene induction. Recently Gadd45 was suggested to be involved in JNK activation. However, as this suggestion relied onin vitro experiments and ectopic overexpression of Gadd45 protein, we examined whether physiological levels of Gadd45 that are induced following exposure to DNA damaging agents and stress can lead to JNK induction. We found that JNK activation by UV irradiation and anisomycin treatment precedes the induction of gadd45mRNA by these agents. Gadd45 protein induction by methyl methanesulfonate also lagged behind JNK activation. The use of protein synthesis inhibitors suggested that newly synthesized proteins, including the stress-induced Gadd45, make only a marginal contribution to JNK activation. We also found that stresses such as γ irradiation induce Gadd45 and do not activate JNK in mouse fibroblasts. Therefore, stress-induced JNK does not depend on Gadd45 induction.

Resistance to p53-mediated growth arrest and apoptosis in Hep 3B hepatoma cells

Mutations in the tumor suppressor p53 are a common event in hepatocellular carcinoma (HCC). Because HCCs typically occur in livers with chronic injury and impaired function, we have explored the role of wild-type p53 in regulating the growth and differentiation of Hep 3B hepatoma cells, a p53-negative line derived from a liver cancer. Stable Hep 3B cell lines were generated in which inducible p53 was introduced using either a temperature-sensitive mutant (p53val135) or a tamoxifen-regulated p53-estrogen receptor chimera (p53-mERtm-pBabepuro). In both cell lines, induction of transcriptionally active p53 was confirmed by assessing several p53 targets: Mdm2 protein, p21waf1 mRNA and protein, and the cyclin G promoter. Despite marked induction of p21waf1, cells with active p53 failed to undergo growth arrest, which is probably due to the presence of a non-functional retinoblastoma protein (pRb) in these cells. Apoptosis also was not observed, even after prolonged (48 h) serum starvation or exposure to cisplatinum. Lack of apoptosis was correlated with unchanged bax mRNA levels following p53 induction. Additionally, albumin mRNA levels remained unchanged, and there was no change in basal transactivation of a reporter containing the promoter of the haptoglobin gene, encoding an acute phase protein. This suggests that growth arrest may be required to promote liver-specific gene expression. Overall, our data demonstrate that introduction of transcriptionally active p53 does not alter the malignant, dedifferentiated phenotype of Hep 3B hepatoma cells. Hence, not all cancer cells are equally responsive to the re-activation of wild-type 53. The ability of a cancer cell to undergo p53-mediated phenotypic alterations may depend on the retention of functional downstream effector pathways.

Fbw7 regulates the activity of endoreduplication mediators and the p53 pathway to prevent drug-induced polyploidy

Fbw7 is a tumor suppressor that is mutated in numerous cancers. It encodes an E3 ubiquitin ligase, whose ability to decrease the levels of pivotal regulators of cell growth and proliferation underlies its tumor suppressor function. Here, we explore the consequences of Fbw7 inactivation on the outcome of chemotherapeutic treatments. When exposed to spindle toxins such as vinblastine and taxol, Fbw7-deficient cells undergo extensive mitotic slippage and endoreduplication, rendering them polyploid. A combined deregulation of several Fbw7 target proteins is required for this phenotype. Specifically, elevated expression of cyclin E and Aurora A in Fbw7-deficient cells is required for drug-induced polyploidy. However, overexpression of either cyclin E or Aurora A alone is not sufficient for drug-induced polyploidy. In addition, we demonstrate that Fbw7 deficiency limits the ability of p53 to respond to mitotic toxins but not to DNA damage. Furthermore, Fbw7 expression regulates the p53-dependent induction of genes such as Lats2 and p21 in response to vinblastine. Hence, we suggest that Fbw7 serves as a master regulator of the mitotic and tetraploidy checkpoints.

Induction of transcriptionally active jun proteins regulates drug-induced senescence

The drug hydroxyurea (HU) is used for cancer therapy and treatment of sickle cell anemia. It inhibits cell cycle progression by blocking DNA synthesis and drives cells to undergo apoptosis or enter senescence. We demonstrate here that HU induces the expression of two AP-1 proteins, c-Jun and JunB, which exert antagonistic effects on the cell cycle. Moreover, the induction of c-Jun is observed following treatment with two other drugs that inhibit the cell cycle in S phase, aphidicolin and camptothecin. The induction of c-Jun, which promotes cell cycle progression, up-regulates expression of cyclin D after exposure of cells to HU. Deficiency in c-jun prevents elevation of cyclin D expression and extends entrance into HU-induced senescence but also renders cells more resistant to HU-dependent apoptosis. The induction of c-Jun is independent of JNK activity, and additionally, of c-Jun autoregulatory activity but is inhibited upon inhibition of protein kinase C activity. Therefore, we suggest that c-Jun activity prevents drug-induced senescence. Conversely, the JunB target gene, tumor suppressor p16INK4a, a cyclin-dependent kinase inhibitor essential for the induction of drug-induced senescence, is also up-regulated by HU in a JunB-dependent manner. Constitutive expression of JunB up-regulates p16INK4a and increases the sensitivity of mouse fibroblasts to drug-induced-senescence. Thus, we suggest that in contrast to c-Jun, JunB drives cells to enter HU-dependent senescence. The effect of HU treatment, which regulates the intricate web of AP-1 transcription, depends on the balance between c-Jun and JunB activities.

Jun proteins inhibit autophagy and induce cell death

Starvation induces a vigorous autophagic response to enhance cellular survival, whereas nutrient and serum supplementation inhibit autophagy and induce an intensive transcriptional burst that enables cellular proliferation. We recently found that some of the genes induced by serum and growth factors—the immediate early proteins JunB and c-Jun—inhibit autophagy. Deregulation of JunB expression when autophagy is specifically required, tilts the fate of starved cells to apoptosis.

P53-Mediated Apoptosis

The p53 gene is an important tumor suppressor gene, whose inactivation appears to play a pivotal role in many types of cancer1–3. The p53 protein, encoded by this gene, is a potent sequence-specific transcriptional activator4,5. Binding of the p53 protein to genes which contain consensus p53 binding sites results in a pronounced increase in their transcription rates6–11. Transcriptional activation of relevant target genes, mediated through high affinity sequence-specific DNA binding, is believed to be responsible for many of the biological effects of the wild-type p53 (wt p53) protein. This is supported by the fact that the overwhelming majority of p53 mutations found in human tumors abrogate DNA binding, either by altering direct DNA contact residues or through destabilizing the structure of the DNA binding domain12,13.

767 The role of p53 in the control of cell death

The rate of growth of a cell population is dictated by the balance between proliferation on the one hand, and terminal differentiation and cell death on the other hand. Genes controlling either of these processes are likely to affect cancer development. In line with this notion, the p53 tumor suppressor gene was found to be a positive regulator of cell death. In certain cancer cells, reactivation of functional wild type (wt) p53 can trigger cell death, with distinctive features of apoptosis. Furthermore, wt p53 is required in many cases for the efficient induction of apoptosis by DNA damage or by deprivation of survival factors. We have developed an assay for the analysis of apoptosis in trans-fected HeLa cells. Using this assay, it was shown that wt p53, but not tumor-derived p53 mutants, can serve as a potent inducer of apoptosis. The pRB, the product of another tumor suppressor, can protect HeLa cells from p53-mediated apoptosis. This suggests an inverse relationship between certain growth inhibitory signals and cell death. Further experiments in the HeLa assay suggest that p53, despite being primarily a transcriptional activator, can induce apoptosis without the need to activate target genes. The relationship of these findings to the tumor suppressor effects of p53 will be discussed.