Anti-tumorigenic effects of Type 1 interferon are subdued by integrated stress responses.

Viral and pharmacological inducers of protein kinase RNA-activated (PKR)-like ER kinase (PERK) were shown to accelerate the phosphorylation-dependent degradation of the IFNAR1 chain of the Type 1 interferon (IFN) receptor and to limit cell sensitivity to IFN. Here we report that hypoxia can elicit these effects in a PERK-dependent manner. The altered fate of IFNAR1 affected by signaling downstream of PERK depends on phosphorylation of eIF2α (eukaryotic translational initiation factor 2-α) and ensuing activation of p38α kinase. Activators of other eIF2α kinases such as PKR or GCN2 (general control nonrepressed-2) are also capable of eliminating IFNAR1 and blunting IFN responses. Modulation of constitutive PKR activity in human breast cancer cells stabilizes IFNAR1 and sensitizes these cells to IFNAR1-dependent anti-tumorigenic effects. Although downregulation of IFNAR1 and impaired IFNAR1 signaling can be elicited in response to amino-acid deficit, the knockdown of GCN2 in melanoma cells reverses these phenotypes. We propose that, in cancer cells and the tumor microenvironment, activation of diverse eIF2α kinases followed by IFNAR1 downregulation enables multiple cellular components of tumor tissue to evade the direct and indirect anti-tumorigenic effects of Type 1 IFN.

PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

To proliferate and expand in an environment with limited nutrients, cancer cells co-opt cellular regulatory pathways that facilitate adaptation and thereby maintain tumor growth and survival potential. The endoplasmic reticulum (ER) is uniquely positioned to sense nutrient deprivation stress and subsequently engage signaling pathways that promote adaptive strategies. As such, components of the ER stress-signaling pathway represent potential antineoplastic targets. However, recent investigations into the role of the ER resident protein kinase, RNA-dependent protein kinase (PKR)-like ER kinase (PERK) have paradoxically suggested both pro- and anti-tumorigenic properties. We have used animal models of mammary carcinoma to interrogate the contribution of PERK in the neoplastic process. The ablation of PERK in tumor cells resulted in impaired regeneration of intracellular antioxidants and accumulation of reactive oxygen species triggering oxidative DNA damage. Ultimately, PERK deficiency impeded progression through the cell cycle because of the activation of the DNA damage checkpoint. Our data reveal that PERK-dependent signaling is used during both tumor initiation and expansion to maintain redox homeostasis, thereby facilitating tumor growth.

1alpha,25-dihydroxyvitamin D(3) (calcitriol) and its analogue, 19-nor-1alpha,25(OH)(2)D(2), potentiate the effects of ionising radiation on human prostate cancer cells.

Radiotherapy with external beam radiation or brachytherapy is an established therapeutic modality for prostate cancer. Approximately 30% of patients with localised prostate cancer relapse at the irradiated site. Secondary effects of ionising radiation (IR), for example, bowel and bladder complications, are common. Thus, the search for biological response modifiers that could potentiate the therapeutic effects of radiation and limit the occurrence of serious side effects is an important task in prostate cancer therapy. 1alpha,25-Dihydroxyvitamin D(3) (calcitriol), the active metabolite of vitamin D, and its analogues are under investigation for the treatment of several malignancies including prostate cancer. Here, we report that 1alpha,25-dihydroxyvitamin D(3) and its less calcaemic analogue 19-nor-1alpha,25-(OH)(2)D(2) (Zemplar) act synergistically with IR to inhibit the growth of the human prostate cancer cells in vitro. 1alpha,25-dihydroxyvitamin D(3) potentiated IR-induced apoptosis of LNCaP cells, and nanomolar doses of 1alpha,25-dihydroxyvitamin D(3) and 19-nor-1alpha,25-(OH)(2)D(2) showed synergistic inhibition of growth of LNCaP cells at radiobiologically relevant doses of IR (1-2 Gy). At higher doses of IR, the combination of 1alpha,25-dihydroxyvitamin D(3) and IR or 19-nor-1alpha,25-(OH)(2)D(2) and IR resulted in moderate antagonism. The synergistic effect at radiobiologically relevant doses of radiation suggests that a combination of 1alpha,25-dihydroxyvitamin D(3) or 19-nor-1alpha,25-(OH)(2)D(2) with IR could permit a reduction in the dose of radiation given clinically and thus potentially reduce treatment-related morbidity.

Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation.

Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.

p53 promotes selection for Fas-mediated apoptotic resistance.

Although p53 inactivation is implicated as a mechanism to explain diminished apoptotic response, it is clear that tumor cells that possess transcriptionally functional p53 can also be resistant to diverse apoptotic stimuli. We hypothesize that oncogenic activation and DNA damage are sufficient stimuli to increase the p53-dependent transcription of Fas and thereby establish a situation in which cell to cell contact could be a selective pressure to either lose p53 function or inactivate components of the Fas death pathway. Examination of genetically matched tumor cell lines that possessed either wild-type or null p53 loci indicated that cells possessing functional p53 increased their surface levels of Fas and Fas ligand (FasL) in response to DNA damage. In contrast, stress induced by changes in the tumor microenvironment such as decreased oxygen did not up-regulate Fas or FasL. Cells with wild-type p53 underwent Fas-mediated killing in the presence of either FasL-expressing killer cells or activating Fas antibodies, whereas cells in which p53 was deleted or inactivated were protected from such killing. Furthermore, Fas and FasL expression and induction became transcriptionally repressed in transformed cells with wild-type p53 with increasing passage, whereas other p53 downstream targets and functions, such as p21 inducibility and cell cycle arrest, remained intact. Repression of the Fas locus could be reverted by treatment with the histone deacetylase inhibitor trichostatin A. These results support a model of tumor progression in which oncogenic transformation drives tumor cells to lose either p53 or their Fas sensitivity as a means of promoting their survival and evade immune surveillance.

Hypoxia induces p53 accumulation through MDM2 down-regulation and inhibition of E6-mediated degradation.

Hypoxia, a result of DNA-damaging agents such as ionizing radiation, induces the nuclear accumulation of the p53 tumor suppressor protein. However, unlike the effect in ionizing radiation, hypoxia readily induces the nuclear accumulation of p53 in HPV E6-infected cells. In HPV-infected cells, a key regulator of p53 protein levels is the E6 oncoprotein. In association with the endogenous cellular protein E6-associated protein (E6AP), E6 can accelerate the degradation of p53 under aerobic conditions. To better define the mechanism of p53 induction in E6-infected cells by hypoxia, we studied the expression and association of E6 and E6AP with p53 in vivo. We found that hypoxia did not alter the protein levels of E6 or E6AP as compared with those found under aerobic growth conditions, indicating that protein inhibition of E6 or E6AP alone is not sufficient to explain the increased accumulation of p53 under hypoxic conditions. However, p53 did fail to coprecipitate with E6AP under hypoxia, indicating that hypoxia uncouples the interaction of p53 with E6 and E6AP. We also present evidence to indicate that hypoxia decreases the expression of the endogenous cellular regulator of p53 protein, the human MDM2 protein, resulting in an inhibition of p53 export from the nucleus to the cytoplasm for degradation. Taken together, these results suggest that the hypoxic induction of p53 is attributable to the down-regulation of MDM2 protein levels and uncoupling of p53 from its interaction with the E6/E6AP complex.

Transformed cells require continuous activity of RNA polymerase II to resist oncogene-induced apoptosis.

Studies have indicated that deregulated oncogene expression can result in either programmed cell death or proliferation, depending on the cellular microenvironment. However, little is known about whether oncogenic signals in themselves are able to activate a cellular apoptotic program. We have tested the hypothesis that oncogenic signals in the absence of gene expression are sufficient to induce cell death, which would indicate that constitutive expression of antiapoptotic genes is necessary for maintenance of the transformed state. Using two highly specific RNA polymerase (RNAP) II inhibitors, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin, which inhibit RNAP II function by two distinct mechanisms, we found that inhibition of gene expression substantially increased apoptosis in a time- and dose-dependent manner in p53+/+- and p53(-/-)-transformed mouse embryonic fibroblasts and in HeLa cells, demonstrating that this type of apoptosis does not require wild-type p53. Engineered expression of an alpha-amanitin resistance RNAP II gene rendered cells resistant to induction of apoptosis by alpha-amanitin without affecting their sensitivity to DRB, indicating that alpha-amanitin induces apoptosis solely by inhibiting RNAP II function and not by a nonspecific mechanism. DRB-induced apoptosis was independent of the cell cycle or ongoing DNA replication, since DRB induced similar levels of apoptosis in asynchronous cells and cells synchronized by collection at mitosis. Inhibition of RNAP II in untransformed cells like Rat-1 or human AG1522 fibroblasts resulted not in apoptosis but in growth arrest. In contrast, deregulated expression of c-Myc in Rat-1 cells dramatically increased their sensitivity to DRB, directly demonstrating that apoptosis following inhibition of RNAP II function is greatly enhanced by oncogenic expression. The requirement for RNAP II function to prevent oncogene-induced apoptosis implies the need for the constitutive expression of an antiapoptotic gene(s) to maintain the transformed state. The differential sensitivities of untransformed and transformed cells to induction of apoptosis by transcriptional inhibition, coupled with the finding that this type of apoptosis is independent of p53 status, suggest that inhibition of RNAP II may be exploited therapeutically for the design of successful antitumor agents.

The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: replication protein A hyperphosphorylation and p53 induction.

Severe combined immunodeficient (SCID) mice display an increased sensitivity to ionizing radiation compared with the parental, C.B-17, strain due to a deficiency in DNA double-strand break repair. The catalytic subunit of DNA-dependent protein kinase (DNA-PKCS) has previously been identified as a strong candidate for the SCID gene. DNA-PK phosphorylates many proteins in vitro, including p53 and replication protein A (RPA), two proteins involved in the response of cells of DNA damage. To determine whether p53 and RPA are also substrates of DNA-PK in vivo following DNA damage, we compared the response of SCID and MO59J (human DNA-PKcs-deficient glioblastoma) cells with their respective wild-type parents following ionizing radiation. Our findings indicate that (i) p53 levels are increased in SCID cells following ionizing radiation, and (ii) RPA p34 is hyperphosphorylated in both SCID cells and MO59J cells following ionizing radiation. The hyperphosphorylation of RPA p34 in vivo is concordant with a decrease in the binding of RPA to single-stranded DNA in crude extracts derived from both C.B-17 and SCID cells. These results suggest that DNA-PK is not the only kinase capable of phosphorylating RPA. We conclude that the DNA damage response involving p53 and RPA is not associated with the defect in DNA repair in SCID cells and that the physiological substrate(s) for DNA-PK essential for DNA repair has not yet been identified.

The use of a reversible transcription inhibitor, DRB, to investigate the involvement of specific proteins in the ocular circadian system of Aplysia.

Previously, the effects of 2-h treatments with the reversible transcription inhibitor 5,6-dichloro-1-beta-D-ribobenzimidazole (DRB) on the phase of the circadian rhythm in the eye of Aplysia californica were studied. Here we report a study of the effects of DRB on protein synthesis and a more detailed investigation of the effects of DRB on the phase of the circadian rhythm. Treatments of DRB for 30 min reduced the rate of transcription to about 30% of control values, and this inhibition reversed completely within 2 h after the end of the treatment. A phase-response curve was obtained for 30-min treatments of DRB. Shorter (30 min) treatments with DRB produced phase shifts comparable to those produced by treatments with DRB for 2 h. The phase-response curve obtained using 30-min treatments of DRB was similar to one obtained using 2-h treatments with respect to the phase at which DRB exerts its maximum effect on the rhythm (around circadian time [CT] 6). However, some aspects of the two phase-response curves were different. The effect of DRB on the phase of the rhythm appeared rapidly after removal of DRB treatments given during CT 22-6, but the effects of DRB on the phase of the rhythm appeared more slowly (approximately 10 h) after the treatments given during CT 6-12. Because the effects of DRB on the phase of the overt rhythm appear to be rapid at a particular phase, it is very likely that DRB affects the phase of the rhythm by altering the synthesis of proteins during or shortly after the treatment. Thus we searched for proteins whose synthesis was altered by DRB. Incorporation of labeled amino acids into 2 proteins was found to be altered during the DRB treatment, whereas 15 proteins were affected after the DRB treatment. Among the proteins affected during or shortly after the DRB treatment were four previously identified proteins affected by other treatments that can shift the phase of the eye circadian rhythm. These four proteins are worthy of further study as possible candidates for components of the circadian oscillator.

Attenuated response of p53 and p21 in primary cultures of human prostatic epithelial cells exposed to DNA-damaging agents.

The multifocal origin of prostate cancer suggests a pan-organ defect in a tumor suppressor pathway. Although structural mutations in the p53 gene have been implicated in late-stage prostate cancer, little is known about the p53 response to genotoxic stress in normal human prostatic epithelial cells from which adenocarcinomas originate. We found that the majority (10 of 12) of epithelial cell cultures derived from histologically normal tissues of radical prostatectomy specimens failed to exhibit p53 accumulation in response to ionizing radiation. Epithelial cell cultures derived from benign prostatic hyperplasia and a primary prostatic adenocarcinoma also failed to accumulate p53 in response to ionizing radiation. In contrast, cultures of prostatic stromal cells derived from normal, benign prostatic hyperplasia, or adenocarcinoma tissues exhibited a 3-9-fold induction of p53 within 1-3 h after irradiation. Since p53 regulates a cell cycle checkpoint through the induction of the cyclin-cdk inhibitor p21, we examined p21 accumulation and cell cycle arrest following exposure to ionizing radiation. With one exception, epithelial cells that did not display increased p53 or p21 induction did not demonstrate a significant G1-S arrest in response to ionizing radiation, whereas stromal cells that accumulated p53 and p21 exhibited a large cell cycle arrest. These results indicate a functional difference between the DNA damage response of epithelial and stromal prostatic cells and suggest a possible mechanism for the increased susceptibility of prostatic epithelial cells to accumulate genetic alterations.

Identification of three proteins in the eye of Aplysia, whose synthesis is altered by serotonin (5-HT). Possible involvement of these proteins in the ocular circadian system.

Previous results using translation inhibitors in the ocular circadian system of Aplysia suggest that protein synthesis may be involved in the light and serotonin (5-HT) entrainment pathways or perhaps in the circadian oscillator. Proteins have been previously identified whose synthesis was altered by treatments of light capable of perturbing the phase of the circadian rhythm in the eye of Aplysia. We extended these studies by investigating the effects of other treatments that perturb the ocular circadian rhythm on protein synthesis. 5-HT altered the synthesis of nine proteins. Interestingly, five of the proteins affected by treatments with 5-HT were previously shown to be affected by treatments with light. Four of the proteins affected by treatments with 5-HT were also affected by treatments with analogs of cAMP, a treatment which mimics the effects of 5-HT on the ocular circadian rhythm. To identify the cellular function of some of these proteins, we obtained their partial amino acid sequences. Based on these sequences and additional characterizations, a 78-kDa, pI 5.6 Aplysia protein appears to be glucose-regulated protein 78/binding protein, and a 36-kDa, pI 5.7 Aplysia protein appears to be porin/voltage-dependent anion channel. Heat shock experiments on Aplysia eyes revealed that yet another one of the Aplysia proteins (70 kDa) affected by 5-HT appears to be a heat-inducible member (heat shock protein 70) of the family of heat shock proteins. These findings suggest that these three identified proteins, together or individually, may be involved in some way in the regulation of the timing of the circadian oscillator in the eye of Aplysia.

Effects on protein synthesis produced by pairing depolarization with serotonin, an analogue of associative learning in Aplysia.

A form of associative plasticity in Aplysia, activity-dependent neuromodulation, involves the convergence of neuronal activity and the effects of a modulatory transmitter. To investigate the role of protein synthesis in associative plasticity, we examined the effects of a biochemical analogue of activity-dependent neuromodulation on the level of incorporation of labeled amino acid into proteins. To mimic associative training, abdominal ganglia were exposed to paired treatments of a depolarizing agent, elevated potassium, and a modulatory transmitter, serotonin. The effects of elevated potassium and serotonin applied alone were also examined. At least two proteins (nos. 9 and 17) were affected in a nonadditive way by the paired procedure. Incorporation of label into protein 9 was increased by the paired procedure but was not affected by either elevated potassium or serotonin. Incorporation of label into protein 17 was significantly affected by elevated potassium or serotonin, but the effect of the paired procedure was significantly less than the summed effects of elevated potassium and serotonin applied alone. These results indicate that changes in protein synthesis may be important in the induction of associative plasticities. Amino acid sequences of two peptides derived from protein 9 were obtained. Then, a partial cDNA clone for protein 9 was obtained by performing PCR with degenerate primers corresponding to portions of the sequences of the two peptides. The sequence of protein 9 is related to sequences previously reported for a family of genes comprising the stringent starvation protein of Escherichia coli, auxin-induced proteins of plants, and glutathione S-transferases of a number of organisms.

The hunt for mechanisms of circadian timing in the eye of Aplysia.

The goals of our research are to understand how circadian oscillations in the eye of Aplysia california are generated and how entraining agents regulate these oscillations. These goals require identification of the molecular components of the oscillator and entrainment pathways as well as elucidation of the biochemical processes by which these components interact with one another. Our experimental strategy entails tracing environmental information along an entrainment pathway until the last component of the pathway is reached. The isolated eye of Aplysia exhibits a circadian rhythm of optic nerve impulses. This rhythm is regulated by at least two entrainment pathways. A photic pathway entrains the rhythm to light-dark cycles and an efferent serotonergic pathway relays neural information from the CNS to the oscillator. Phase shifting by light appears to involve an increase in the levels of cGMP, depolarization, and protein synthesis. Phase shifting by serotonin appears to involve an increase in the levels of cAMP, hyperpolarization, and protein synthesis. The involvement of protein synthesis in the entrainment pathways, together with the findings that brief treatments of inhibitors of protein synthesis phase shift the rhythm and that continuous treatments of these inhibitors alter the period of the rhythm, indicates that translation is part of the oscillator mechanism. Recent evidence indicates that transcription may also be part of the oscillator mechanism. Brief treatments with DRB, a reversible transcription inhibitor, phase shift the rhythm while continuous treatments with DRB lengthen the period of the rhythm. A comparison of the effects of transcription and translation inhibitors on the rhythm indicates that transcription and translation are closely coupled in the eye circadian system. To know the precise role of transcription and translation in the circadian system, it is necessary to identify and then study specific proteins and mRNAs important for circadian timing. To identify putative oscillator proteins (POPs), we have hunted for proteins whose synthesis or phosphorylation was altered by the entraining agents light and 5-HT and by other agents that perturb the circadian rhythm. By exposing eyes to labeled amino acids in the presence of phase-shifting treatments and then using two-dimensional gel electrophoresis to separate proteins, we found eight proteins that may be considered POPs. To elucidate the cellular function of POPs, we have begun to obtain their amino acid sequences. A 40,000, pI 5.6 protein (POP-1) was identified as a member of the lipocortin family of proteins. Lipocortins are Ca(2+)-phospholipid binding proteins whose functions include inhibition of PLA2.(ABSTRACT TRUNCATED AT 400 WORDS)

Alteration of the phase and period of a circadian oscillator by a reversible transcription inhibitor.

A function for transcription in the mechanism of a circadian oscillator was investigated with the reversible transcription inhibitor 5,6-dichloro-1-beta-D- ribobenzimidazole (DRB). Two-hour treatments with DRB shifted the phase of the circadian rhythm of the isolated eye of Aplysia, and continuous treatments of DRB lengthened the free running period of this rhythm. Camptothecin, an inhibitor of transcription that is structurally unrelated to DRB, had similar effects on the circadian rhythm. These results suggest that transcription may be part of the circadian oscillating mechanism.