Medial Temporal Lobe Contributions to Episodic Future Thinking: Scene Construction or Future Projection?

Previous research has shown that the medial temporal lobes (MTL) are more strongly engaged when individuals think about the future than about the present, leading to the suggestion that future projection drives MTL engagement. However, future thinking tasks often involve scene processing, leaving open the alternative possibility that scene-construction demands, rather than future projection, are responsible for the MTL differences observed in prior work. This study explores this alternative account. Using functional magnetic resonance imaging, we directly contrasted MTL activity in 1) high scene-construction and low scene-construction imagination conditions matched in future thinking demands and 2) future-oriented and present-oriented imagination conditions matched in scene-construction demands. Consistent with the alternative account, the MTL was more active for the high versus low scene-construction condition. By contrast, MTL differences were not observed when comparing the future versus present conditions. Moreover, the magnitude of MTL activation was associated with the extent to which participants imagined a scene but was not associated with the extent to which participants thought about the future. These findings help disambiguate which component processes of imagination specifically involve the MTL.

Using future thinking to reduce temporal discounting: Under what circumstances are the medial temporal lobes critical?

The capacity to envision the future plays an important role in many aspects of cognition, including our ability to make optimal, adaptive choices. Past work has shown that the medial temporal lobe (MTL) is necessary for decisions that draw on episodic future thinking. By contrast, little is known about the role of the MTL in decisions that draw on semantic future thinking. Accordingly, the present study investigated whether the MTL contributes to one form of decision making, namely intertemporal choice, when such decisions depend on semantic consideration of the future. In an intertemporal choice task, participants must select either a smaller amount of money that is available in the present or a larger amount of money that would be available at a future date. Amnesic individuals with MTL damage and healthy control participants performed such a task in which, prior to making a choice, they engaged in a semantic generation exercise, wherein they generated items that they would purchase with the future reward. In experiment 1, we found that, relative to a baseline condition involving standard intertemporal choice, healthy individuals were more inclined to select a larger, later reward over a smaller, present reward after engaging in semantic future thinking. By contrast, amnesic participants were paradoxically less inclined to wait for a future reward following semantic future thinking. This finding suggests that amnesics may have had difficulty "tagging" the generated item(s) as belonging to the future. Critically, experiment 2 showed that when the generated items were presented alongside the intertemporal choices, both controls and amnesic participants shifted to more patient choices. These findings suggest that the MTL is not needed for making optimal decisions that draw on semantic future thinking as long as scaffolding is provided to support accurate time tagging. Together, these findings stand to better clarify the role of the MTL in decision making.

Does the hippocampus keep track of time?

In the present study, we examined the role of the medial temporal lobe (MTL) in prospective time estimation at short and long timescales using a novel behavioral paradigm adapted from rodent work. Amnesic patients with MTL damage and healthy control participants estimated the duration of nature-based video clips that were either short (≤ 90 s) or long (more than 4 min). Consistent with previous work in rodents, we found that amnesic patients were impaired at making estimations for long, but not for short durations. Critically, these effects were observed in patients who had lesions circumscribed to the hippocampus, suggesting that the pattern observed was not attributable to the involvement of extra-hippocampal structures. That the MTL, and more specifically the hippocampus, is critical for prospective temporal estimation only at long intervals suggests that multiple neurobiological mechanisms support prospective time estimation.

The effects of adding zoledronic acid to neoadjuvant chemotherapy on tumour response: exploratory evidence for direct anti-tumour activity in breast cancer.

BACKGROUND: Pre-clinical studies have demonstrated synergistic anti-tumour effects of chemotherapy (CT) and zoledronic acid (ZOL). Within the AZURE trial, designed to determine whether the addition of ZOL to neoadjuvant therapy improves disease outcomes, a subgroup received neoadjuvant CT. We report a retrospective evaluation comparing pathological response in the primary tumour between treatment groups. METHODS: In total, 205 patients received neoadjuvant CT+/-ZOL (CT+ZOL, n=102; CT, n=103). The primary end point was pathologically assessed residual invasive tumour size (RITS) at surgery. Secondary end points were pathological complete response (pCR) rate and axillary nodal involvement. Following review of surgical pathology reports (n=195), outcome differences between groups were assessed adjusting for potential response modifiers. RESULTS: Baseline characteristics and CT treatments were similar. In multivariate analysis, allowing for biological and clinical factors known to influence tumour response, the adjusted mean RITS in CT and CT+ZOL groups were 27.4 and 15.5 mm, respectively, giving a difference in means of 12 mm (95% confidence interval: 3.5-20.4 mm; P=0.006). The pCR rate was 6.9% in the CT group and 11.7% in the CT+ZOL group (P=0.146). There was no difference in axillary nodal involvement (P=0.6315). CONCLUSION: These data suggest a possible direct anti-tumour effect of ZOL in combination with CT, warranting formal evaluation in prospective studies.

Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2.

We investigated whether combined treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and trastuzumab could enhance the specific killing of cells that overexpress the erbB-2 receptor. The combination resulted in an enhancement of TRAIL-mediated apoptosis in all cell lines overexpressing erbB-2 receptor compared with either reagent alone. In contrast, there was no effect in cell lines with low levels of the erb-B2 receptor. Trastuzumab treatment resulted in down-regulation of the erbB-2 receptor in all erbB-2-overexpressing cell lines. Similar enhancement of TRAIL toxicity was observed when the erbB-2 receptor was down-regulated using antisense oligodeoxynucleotides. Down-regulation of the erbB-2 receptor protein by trastuzumab or antisense oligodeoxynucleotides decreased Akt kinase activation but not mitogen-activated protein kinase activation. Down-regulation of Akt kinase activity by a phosphatidylinositol 3'-kinase inhibitor (LY294002) also resulted in enhancement of TRAIL-mediated apoptosis. Expression of a constitutively active form of Akt kinase in an erbB-2-overexpressing cell line completely abrogated the increase in TRAIL-mediated apoptosis by trastuzumab and significantly reduced the biological effect of either reagent alone. Therefore, down-regulation of the erbB-2 receptor by trastuzumab enhances TRAIL-mediated apoptosis by inhibiting Akt kinase activity. These data suggest that the combination of trastuzumab and TRAIL may allow enhanced therapeutic efficacy and specificity in the treatment of erbB-2-overexpressing tumors.

Recognition memory in amnesia: effects of relaxing response criteria.

In two experiments, using the remember/know paradigm, we examined whether recognition memory in amnesic patients can be improved by instructing patients to relax their response criterion. Experiment 1 was modeled after a study by Dorfman, Kihlstrom, Cork, and Misiaszek (1995), in which direct instructions to respond more leniently led to an increase in recognition accuracy in patients with ECT-induced amnesia. We failed to extend this finding to patients with global amnesia, but the manipulation was unsuccessful in control subjects as well. In Experiment 2, response criterion was manipulated indirectly by providing information about the alleged base rate of study items on the recognition test. This manipulation led to a criterion shift in control subjects and enhanced discriminability in amnesic patients. Analysis of "remember" and "know" responses suggests that improved accuracy in amnesia was associated with enhanced familiarity-based recognition.

The role of explicit memory processes in cross-modal priming: an investigation of stem completion priming in amnesia.

To clarify the role of explicit memory processes in cross-modal priming, two experiments examined the status of cross-modal stem completion priming in amnesia. Experiment 1 used a standard behavioral paradigm in which stems corresponding to studied and unstudied words were intermixed. Amnesic patients showed intact within- and cross-modal priming, but, in contrast to controls, they recognized very few of their completions as having been on the study list. This finding suggests that memorial awareness is not necessary for cross-modal priming to occur. Experiment 2 used a paradigm modeled after functional imaging studies, in which stems corresponding to studied and unstudied words were blocked. Amnesic patients showed intact within-modal priming, but impaired cross-modal priming. This finding is consistent with the notion that a blocked format induces voluntary retrieval strategies in normal participants.

Preserved priming in auditory perceptual identification in Alzheimer's disease.

To examine the status of auditory perceptual priming in Alzheimer's disease (AD), this study examined the performance of AD patients in auditory perceptual identification of words. In Experiment 1, the processing operations required to perform the tasks at study and test were matched, whereas in Experiment 2, processing operations at study and test were mismatched. AD patients showed normal priming in both experiments, despite impaired recognition memory. These findings extend to the auditory domain the finding of intact perceptual implicit memory in AD. Preserved auditory priming in AD may reflect the operation of a pre-semantic, phonological representation system, localized to posterior neocortical areas that are functionally spared in AD.

The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of IkappaB kinase and c-Jun N-terminal kinase.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Apo2 ligand [Apo2L]) is a member of the TNF superfamily and has been shown to have selective antitumor activity. Although it is known that TRAIL (Apo2L) induces apoptosis and activates NF-kappaB and Jun N-terminal kinase (JNK) through receptors such as TRAIL-R1 (DR4) and TRAIL-R2 (DR5), the components of its signaling cascade have not been well defined. In this report, we demonstrated that the death domain kinase RIP is essential for TRAIL-induced IkappaB kinase (IKK) and JNK activation. We found that ectopic expression of the dominant negative mutant RIP, RIP(559-671), blocks TRAIL-induced IKK and JNK activation. In the RIP null fibroblasts, TRAIL failed to activate IKK and only partially activated JNK. The endogenous RIP protein was detected by immunoprecipitation in the TRAIL-R1 complex after TRAIL treatment. More importantly, we found that RIP is not involved in TRAIL-induced apoptosis. In addition, we also demonstrated that the TNF receptor-associated factor 2 (TRAF2) plays little role in TRAIL-induced IKK activation although it is required for TRAIL-mediated JNK activation. These results indicated that the death domain kinase RIP, a key factor in TNF signaling, also plays a pivotal role in TRAIL-induced IKK and JNK activation.

Inhibition of NF-kappaB activity enhances TRAIL mediated apoptosis in breast cancer cell lines.

Most breast cancer cell lines are resistant to TNF-related apoptosis inducing ligand (TRAIL) induced apoptosis. In sensitive breast cancer cell lines TRAIL rapidly induces the cleavage and activation of caspases leading to the subsequent cleavage of downstream caspase substrates. In contrast, there is no caspase activation in the resistant cell lines. The transcription factor NF-KB can inhibit apoptosis induced by a variety of stimuli including activation of death receptors. We investigated whether NF-kappaB contributes to the resistance of breast cancer cells to TRAIL induced apoptosis. All of the resistant breast cancer cell lines expressed NF-kappaB and had detectable NF-kappaB activity in nuclear extracts prior to treatment with TRAIL. Upon TRAIL treatment, a significant increase in NF-kappaB activity was seen in most of the cell lines. To directly test if NF-kappaB activity contributes to the resistance of these cell lines to TRAIL, we transiently transfected the resistant cell lines with an inhibitor of NF-kappaB (IkappaBdeltaN) and measured TRAIL induced apoptosis in control and transfected cells. All of the resistant cell lines tested showed an increase in TRAIL induced apoptosis when transfected with the IKBdeltaN. These results demonstrate that TRAIL resistant breast cancer cells fail to rapidly activate the apoptotic machinery but they do activate NF-kappaB. Inhibition of NF-kappaB activity increases the sensitivity to TRAIL mediated apoptosis in resistant cells. These results suggest that agents which inhibit NF-kappaB should increase the clinical efficacy of TRAIL in breast cancer cells.

The 100-kDa proteolytic fragment of RB is retained predominantly within the nuclear compartment of apoptotic cells.

The retinoblastoma tumor suppressor protein (RB) has been shown to play a role in regulating the eukaryotic cell cycle, promoting cellular differentiation, and modulating programmed cell death. Although regulation of RB tumor suppressor activity is mediated by reversible phosphorylation, an additional posttranslational modification involves the cleavage of 42 residues from the carboxy terminus of RB during the onset of drug-induced or receptor-mediated apoptosis. We now demonstrate that a recombinant p100cl RB species localizes to the nucleus where it may retain wildtype "pocket" protein binding activity. In addition, using immunocytochemistry, we show that cleavage of the endogenous RB protein occurs in vivo in human cells and that p100cl is predominantly retained within the nuclear compartment of cells during early apoptosis. We also show that the carboxy-terminal cleavage of RB is detected immediately following caspase-3 and PARP cleavage during FAS-mediated apoptosis of MCF10 cells. These findings suggest that this cleavage event may be a component of a downstream cascade during programmed cell death.

cbl-b inhibits EGF-receptor-induced apoptosis by enhancing ubiquitination and degradation of activated receptors.

Studies in C. elegans and Drosophila melanogaster suggest that cbl proteins are inhibitors of epidermal growth factor receptor (EGFR) function. Here we describe that overexpression of cbl-b, a homologue of the c-cbl protooncogene, inhibits EGFR-induced apoptosis in MDA-MB-468 breast cancer cells. Overexpression of cbl-b results in a shortened duration of EGFR activation upon EGF stimulation. This is demonstrated by decreased amounts of phosphorylated EGFR as well as by inhibition of multiple downstream signaling pathways. The inhibition of signaling by cbl-b results from increased ubiquitination and degradation of the activated EGFR. The inhibitory effects of cbl-b overexpression on apoptosis and on EGFR signaling are reversed by blocking proteosomal degradation of the EGFR. These data demonstrate that the mechanism by which cbl-b inhibits EGFR-induced apoptosis is by activation-dependent degradation of the EGFR. They imply that this mechanism may be a general one whereby cbl proteins regulate intracellular signaling.

cbl-3: a new mammalian cbl family protein.

We have cloned a new human gene, cbl-3, which encodes a protein with marked homology to the cbl family of proteins. The predicted protein encoded by this gene retains the conserved phosphotyrosine binding domain (PTB) in the N-terminal and the zinc finger but is significantly shorter (MW 52.5 kDa) than the other mammalian cbl proteins. The protein lacks the extensive proline rich domain and leucine zipper seen in c-cbl and cbl-b and structurally most resembles the C. elegans and Drosophila cbl proteins. The gene is ubiquitously expressed with highest expression in the aerodigestive tract, prostate, adrenal gland, and salivary gland. The protein is phosphorylated and recruited to the EGFR upon EGF stimulation and inhibits EGF stimulated MAP kinase activation. In comparison to the other mammalian cbl proteins (e.g. cbl-b), cbl-3 interacts with a restricted range of proteins containing Src Homology 3 regions. An alternatively spliced form of the cbl-3 protein was also identified which deletes a critical region of the PTB domain and which does not interact with the EGFR nor inhibit EGF stimulated MAP kinase activation. These data demonstrate that cbl-3, a novel mammalian cbl protein, is a regulator of EGFR mediated signal transduction.

cbl-b inhibits epidermal growth factor receptor signaling.

The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.

Chemotherapy augments TRAIL-induced apoptosis in breast cell lines.

Expression and function of the TRAIL apoptotic pathway was investigated in normal and malignant breast epithelial cells. Glutathione-S-transferase (GST)-TRAIL extracellular domain fusion proteins were produced to analyze TRAIL-induced apoptosis. Only GST-TRAIL constructs containing regions homologous to the Fas self-association and ligand binding domains could induce apoptosis. GST-TRAIL induced significant (>90%) apoptosis in just one of eight normal and one of eight malignant breast cell lines. All other lines were relatively resistant to TRAIL-induced apoptosis. Activating TRAIL receptors DR4 and DR5 were expressed in all normal and malignant breast cell lines. The inhibitory receptor TRID was highly expressed in one of four normal and two of seven malignant breast cell lines. DR4, DR5, or TRID expression did not correlate with sensitivity to TRAIL-induced apoptosis. Incubation of cell lines with doxorubicin or 5-fluorouracil significantly augmented TRAIL-induced apoptosis in most breast cell lines. By fractional inhibition analysis, the toxicity of the combination of TRAIL and doxorubicin or 5-fluorouracil was synergistic compared with either agent alone. In contrast, melphalan and paclitaxel augmented TRAIL-induced apoptosis in few cell lines, and methotrexate did not augment it in any cell line. Augmentation of TRAIL-induced apoptosis by doxorubicin or 5-fluorouracil was mediated through caspase activation. This was evidenced by the fact that chemotherapy agents that synergized with TRAIL (e.g., doxorubicin) themselves caused cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and their toxicity was blocked by the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2 (ZVAD-fmk). The combination of TRAIL and doxorubicin caused significantly greater caspase-3 and PARP cleavage, and the combined toxicity also was inhibited by ZVAD-fmk. In contrast, chemotherapy agents that did not augment TRAIL-induced apoptosis (e.g., methotrexate) caused minimal caspase-3 and PARP cleavage by themselves, and their toxicity was not inhibited by ZVAD-fmk. These drugs also did not increase caspase-3 or PARP cleavage when combined with TRAIL. In summary, few breast cell lines are sensitive to TRAIL-induced apoptosis, and no difference in sensitivity is found between normal and malignant cell lines. Treatment with chemotherapy provides an approach to sensitize breast cancer cells to TRAIL-induced apoptosis.

The neural basis of aware and unaware forms of memory.

Information regarding the nature of phenomenal awareness in memory comes from a direct comparison of explicit and implicit memory tasks. Explicit memory tasks require conscious awareness of a prior episode, whereas implicit memory tasks do not. This paper reviews evidence regarding the neural basis of aware and unaware forms of memory as obtained from patient studies and functional neuroimaging work. These studies suggest the existence of a memory system centered in the medial temporal and frontal lobes that is dedicated to the storage and retrieval of episodes and several neocortical memory systems that are dedicated to the processing and representation of perceptual and semantic information. Different hypotheses are discussed as to how the phenomenal awareness that accompanies episodic memories may arise within the hippocampal-frontal memory system. These views have in common the notion that various forms of information need to be bound together to be retrievable as an aware memory, and that the hippocampus is critical to this binding function.

Intact and impaired conceptual memory processes in amnesia.

To examine the status of conceptual memory processes in amnesia, a conceptual memory task with implicit or explicit task instructions was given to amnesic and control groups. After studying a list of category exemplars, participants saw category labels and were asked to generate as many exemplars as possible (an implicit memory task) or to generate exemplars that had been in the prior study list (an explicit memory task). After incidental deep or shallow encoding of exemplars, amnesic patients showed normal implicit memory performance (priming), a normal levels-of-processing effect on priming, and impaired explicit memory performance. After intentional encoding of exemplars, amnesic patients showed impaired implicit and explicit memory performance. Results suggest that although amnesic patients can show impairments on implicit and explicit conceptual memory tasks, their deficit does not generalize to all conceptual memory tasks.

Evidence for multiple mechanisms of conceptual priming on implicit memory tests.

The authors examined effects of encoding manipulations on 4 conceptual-implicit memory tasks: word-cued association, category-cued association, category verification, and abstract/concrete classification. Study-phase conceptual elaboration enhanced priming for word-cued association with weakly associated words (Experiment 3), and for category-cued association with high- and low-dominance exemplars (Experiments 4 and 5), but did not enhance priming for word-cued association with strongly associated words (Experiments 1 and 2), for category verification with high- and low-dominance exemplars (Experiment 5), or for abstract/concrete classification (Experiment 7). Forms of priming that were unaffected by conceptual elaboration were not mediated by perceptual processes because they were unaffected by study-test modality changes (Experiments 6 and 8). The dissociative effects of conceptual elaboration on conceptual-implicit tasks suggest that at least 2 dissociable mechanisms mediate conceptual priming.

Fas expression and function in normal and malignant breast cell lines.

Expression and function of the Fas apoptotic pathway was investigated in normal and malignant human breast epithelial cells. Nontransformed mammary epithelial cell lines all expressed high levels of Fas mRNA and protein, but only one of seven breast cancer cell lines (T47D) expressed high levels of Fas. Apoptosis was induced in the nontransformed lines when they were incubated with the anti-Fas antibody. However, all of the breast cancer cell lines tested, except T47D, were resistant to Fas-mediated apoptosis. Four of five Fas-resistant breast cancer cell lines became sensitive to Fas-mediated apoptosis upon treatment with IFN-gamma. Fas mRNA increased slightly in both cell lines that became sensitive and in the cell line that remained resistant to Fas-mediated apoptosis upon IFN-gamma treatment. However, the cell surface expression of Fas showed little or no increase in any of the cell lines tested upon IFN-gamma treatment. In contrast to Fas expression, interleukin-1beta-converting enzyme (ICE) expression increased only in the cell lines that became Fas sensitive after IFN-gamma treatment. The importance of ICE and/or ICE-like proteases in Fas-mediated apoptosis in these cells was confirmed by inhibition of Fas-mediated apoptosis by a specific ICE inhibitor, YVAD-cmk. Fas sensitivity was reconstituted in the IFN-gamma-resistant cell line by transfection of ICE into that cell line. Together, these data suggest that down-regulation of Fas and its pathway may be a step in tumor progression and that modulation of Fas expression may provide an approach to inducing apoptosis in breast cancer cells.

The protein tyrosine phosphatase DEP-1 is induced during differentiation and inhibits growth of breast cancer cells.

Sodium butyrate-induced differentiation of breast cancer cell lines was used to identify protein tyrosine phosphatases (PTPs) involved in differentiation and growth inhibition of breast cancer cells. Of 42 PTPs analyzed, 31 were expressed in the ZR75-1 breast cancer cell line. Expression of four PTPs (DEP-1, SAP, PTP gamma, and PAC) was regulated in ZR75-1 cells undergoing differentiation. Expression of two of these PTPs (DEP-1 and SAP) was also regulated in the SKBr-3 cell line undergoing differentiation. In view of its marked induction with differentiation in an estrogen receptor (ER)-positive and an ER-negative breast cancer cell line, DEP-1 was investigated for a role in growth inhibition or induction of differentiation in breast cancer cells. A DEP-1 cDNA construct under control of a constitutively active cytomegalovirus promoter was transfected into the ZR75-1, SKBR-3, and MCF-7 breast cancer cell lines, and resistant colonies were selected with G418. DEP-1 expression inhibited the development of resistant colonies by 3-5-fold in all three lines compared to transfection with vector alone. Three stable MCF-7 cell lines expressing DEP-1 under control of an inducible metallothionein promoter were then established. In these lines, induction of DEP-1 expression inhibited breast cancer cell growth by 5-10-fold. These data describe PTPs expressed and regulated in breast cancer cell lines during differentiation and identify one PTP, DEP-1, that inhibits the growth of breast cancer cells in vitro.