Effects of evening smartphone use on sleep and declarative memory consolidation in male adolescents and young adults.

Exposure to short-wavelength light before bedtime is known to disrupt nocturnal melatonin secretion and can impair subsequent sleep. However, while it has been demonstrated that older adults are less affected by short-wavelength light, there is limited research exploring differences between adolescents and young adults. Furthermore, it remains unclear whether the effects of evening short-wavelength light on sleep architecture extend to sleep-related processes, such as declarative memory consolidation. Here, we recorded polysomnography from 33 male adolescents (15.42 ± 0.97 years) and 35 male young adults (21.51 ± 2.06 years) in a within-subject design during three different nights to investigate the impact of reading for 90 min either on a smartphone with or without a blue-light filter or from a printed book. We measured subjective sleepiness, melatonin secretion, sleep physiology and sleep-dependent memory consolidation. While subjective sleepiness remained unaffected, we observed a significant melatonin attenuation effect in both age groups immediately after reading on the smartphone without a blue-light filter. Interestingly, adolescents fully recovered from the melatonin attenuation in the following 50 min before bedtime, whereas adults still, at bedtime, exhibited significantly reduced melatonin levels. Sleep-dependent memory consolidation and the coupling between sleep spindles and slow oscillations were not affected by short-wavelength light in both age groups. Nevertheless, adults showed a reduction in N3 sleep during the first night quarter. In summary, avoiding smartphone use in the last hour before bedtime is advisable for adolescents and young adults to prevent sleep disturbances. Our research empirically supports general sleep hygiene advice and can inform future recommendations regarding the use of smartphones and other screen-based devices before bedtime.

Spectral Slope and Lempel-Ziv Complexity as Robust Markers of Brain States during Sleep and Wakefulness.

Nonoscillatory measures of brain activity such as the spectral slope and Lempel-Ziv complexity are affected by many neurological disorders and modulated by sleep. A multitude of frequency ranges, particularly a broadband (encompassing the full spectrum) and a narrowband approach, have been used especially for estimating the spectral slope. However, the effects of choosing different frequency ranges have not yet been explored in detail. Here, we evaluated the impact of sleep stage and task engagement (resting, attention, and memory) on slope and complexity in a narrowband (30-45 Hz) and broadband (1-45 Hz) frequency range in 28 healthy male human subjects (21.54 ± 1.90 years) using a within-subject design over 2 weeks with three recording nights and days per subject. We strived to determine how different brain states and frequency ranges affect slope and complexity and how the two measures perform in comparison. In the broadband range, the slope steepened, and complexity decreased continuously from wakefulness to N3 sleep. REM sleep, however, was best discriminated by the narrowband slope. Importantly, slope and complexity also differed between tasks during wakefulness. While narrowband complexity decreased with task engagement, the slope flattened in both frequency ranges. Interestingly, only the narrowband slope was positively correlated with task performance. Our results show that slope and complexity are sensitive indices of brain state variations during wakefulness and sleep. However, the spectral slope yields more information and could be used for a greater variety of research questions than Lempel-Ziv complexity, especially when a narrowband frequency range is used.

Sleep spindle maturity promotes slow oscillation-spindle coupling across child and adolescent development.

The synchronization of canonical fast sleep spindle activity (12.5-16 Hz, adult-like) precisely during the slow oscillation (0.5-1 Hz) up peak is considered an essential feature of adult non-rapid eye movement sleep. However, there is little knowledge on how this well-known coalescence between slow oscillations and sleep spindles develops. Leveraging individualized detection of single events, we first provide a detailed cross-sectional characterization of age-specific patterns of slow and fast sleep spindles, slow oscillations, and their coupling in children and adolescents aged 5-6, 8-11, and 14-18 years, and an adult sample of 20- to 26-year-olds. Critically, based on this, we then investigated how spindle and slow oscillation maturity substantiate age-related differences in their precise orchestration. While the predominant type of fast spindles was development-specific in that it was still nested in a frequency range below the canonical fast spindle range for the majority of children, the well-known slow oscillation-spindle coupling pattern was evident for sleep spindles in the adult-like canonical fast spindle range in all four age groups-but notably less precise in children. To corroborate these findings, we linked personalized measures of fast spindle maturity, which indicate the similarity between the prevailing development-specific and adult-like canonical fast spindles, and slow oscillation maturity, which reflects the extent to which slow oscillations show frontal dominance, with individual slow oscillation-spindle coupling patterns. Importantly, we found that fast spindle maturity was uniquely associated with enhanced slow oscillation-spindle coupling strength and temporal precision across the four age groups. Taken together, our results suggest that the increasing ability to generate adult-like canonical fast sleep spindles actuates precise slow oscillation-spindle coupling patterns from childhood through adolescence and into young adulthood.

Cells in the brain are wired together like an electric circuit that can relay information from one area of the brain to the next. Even when sleeping, the human brain continues to send signals to process information it has encountered during the day. This results in two patterns of electrical activity that define the sleeping brain: slowly repeating waves (or slow oscillations) and rapid bursts of activity known as sleep spindles. Although slow oscillations and sleep spindles are generated in different regions of the brain, they often happen at the same time. This syncing of activity is thought to help different parts of the brain to communicate with each other. Such communication is essential for new memories to become stable and last a long time. In children, slow oscillations and sleep spindles appear together less frequently, suggesting that these co-occurring patterns of electrical activity develop as humans grow into adults. Here, Joechner et al. set out to understand what drives slow oscillations and sleep spindles to start happening at the same time. The team used a technique called electroencephalography (or EEG for short) to study the brain activity of children, teenagers and adults as they slept. This revealed that slow oscillations and sleep spindles occur together less often in children compared to teenagers and adults. Moreover, the slow oscillations and sleep spindles observed in the children had very different physical characteristics to those observed in adults. Further analyses showed that the more similar the children's sleep spindles were to adult spindles, the more consistently they appeared at the same time as the slow oscillations. The findings of Joechner et al. suggest that as children grow up, their sleep spindles become more adult-like, causing the spindles to happen at the same time as slow oscillations more consistently. This indicates that brain circuits that generate sleep spindles may play an essential role in developing successful communication networks in the human brain. In the future, this work may ultimately provide new insights into how age-related changes to the brain contribute to cognitive development, and suggests sleep as a potential intervention target for neurodevelopmental disorders.

An evolutionary conserved division-of-labor between archicortical and neocortical ripples organizes information transfer during sleep.

Systems-level memory consolidation during sleep depends on the temporally precise interplay between cardinal sleep oscillations. Specifically, hippocampal ripples constitute a key substrate of the hippocampal-neocortical dialog underlying memory formation. Recently, it became evident that ripples are not unique to archicortex, but constitute a wide-spread neocortical phenomenon. To date, little is known about the morphological similarities between archi- and neocortical ripples. Moreover, it remains undetermined if neocortical ripples fulfill distinct functional roles. Leveraging intracranial recordings from the human medial temporal lobe (MTL) and neocortex during sleep, our results reveal region-specific functional specializations, albeit a near-uniform morphology. While MTL ripples synchronize the memory network to trigger directional MTL-to-neocortical information flow, neocortical ripples reduce information flow to minimize interference. At the population level, MTL ripples confined population dynamics to a low-dimensional subspace, while neocortical ripples diversified the population response; thus, constituting an effective mechanism to functionally uncouple the MTL-neocortical network. Critically, we replicated the key findings in rodents, where the same division-of-labor between archi- and neocortical ripples was evident. In sum, these results uncover an evolutionary preserved mechanism where the precisely coordinated interplay between MTL and neocortical ripples temporally segregates MTL information transfer from subsequent neocortical processing during sleep.

Slow oscillation-spindle coupling strength predicts real-life gross-motor learning in adolescents and adults.

Previously, we demonstrated that precise temporal coordination between slow oscillations (SOs) and sleep spindles indexes declarative memory network development (Hahn et al., 2020). However, it is unclear whether these findings in the declarative memory domain also apply in the motor memory domain. Here, we compared adolescents and adults learning juggling, a real-life gross-motor task. Juggling performance was impacted by sleep and time of day effects. Critically, we found that improved task proficiency after sleep lead to an attenuation of the learning curve, suggesting a dynamic juggling learning process. We employed individualized cross-frequency coupling analyses to reduce inter- and intragroup variability of oscillatory features. Advancing our previous findings, we identified a more precise SO-spindle coupling in adults compared to adolescents. Importantly, coupling precision over motor areas predicted overnight changes in task proficiency and learning curve, indicating that SO-spindle coupling relates to the dynamic motor learning process. Our results provide first evidence that regionally specific, precisely coupled sleep oscillations support gross-motor learning.

Slow oscillation-spindle coupling predicts enhanced memory formation from childhood to adolescence.

Precise temporal coordination of slow oscillations (SO) and sleep spindles is a fundamental mechanism of sleep-dependent memory consolidation. SO and spindle morphology changes considerably throughout development. Critically, it remains unknown how the precise temporal coordination of these two sleep oscillations develops during brain maturation and whether their synchronization indexes the development of memory networks. Here, we use a longitudinal study design spanning from childhood to adolescence, where participants underwent polysomnography and performed a declarative word-pair learning task. Performance on the memory task was better during adolescence. After disentangling oscillatory components from 1/f activity, we found frequency shifts within SO and spindle frequency bands. Consequently, we devised an individualized cross-frequency coupling approach, which demonstrates that SO-spindle coupling strength increases during maturation. Critically, this increase indicated enhanced memory formation from childhood to adolescence. Our results provide evidence that improved coordination between SOs and spindles indexes the development of sleep-dependent memory networks.

Sleep is essential for consolidating the memories that we made during the day. As we lie asleep, unconscious, our brain is busy processing the day's memories, which travel through three parts of the brain before they are filed away. First, the hippocampus, the part of the brain that stores memories temporarily, replays the memories of the day. Then the reactivated memories pass through the thalamus, a central crossroads in the brain, so they can be embedded in the neocortex for long-term storage. Neuroscientists can eavesdrop on the brain at work, day or night, using a technique called EEG. Short for electroencephalogram, an EEG detects brain waves like the bursts of electrical activity known as sleep spindles and slower sleep waves called slow oscillations. These two brain wave patterns represent how the brain processes memories as people sleep ­ and it is all about timing. If the two patterns are running in sync, then the brain's memory systems are thought to be communicating well and memories are more likely to be stored. But patterns of sleep spindles and slow oscillations change dramatically between childhood and adolescence. Memory consolidation also improves in those formative years. Still, it is not yet known if better synchronization between sleep spindles and slow oscillations explains how memory formation improves during this period; that is the going theory. To test it out, Hahn et al. completed a unique study examining how well a group of 33 children could store memories, and then again when the same group were teenagers. Both times, the group was asked to memorise and then recall a set of words before and after a full night's sleep. Hahn et al. measured how much their memory recall improved and whether their brain wave patterns were in sync, looking for any changes between childhood and adolescence. This showed that children whose sleep spindles stacked better with their slow oscillations had improved memory formation once they became teenagers. This work highlights how communication between memory systems in the brain improves as children age, and so does memory. Moreover, it suggests that if disturbances were to be detected in patterns of sleep spindles and slow oscillations, there might be some problem with memory storage. It also points to brain stimulation as a possible treatment option for such problems in the future.

About the Accuracy and Problems of Consumer Devices in the Assessment of Sleep.

Commercial sleep devices and mobile-phone applications for scoring sleep are gaining ground. In order to provide reliable information about the quantity and/or quality of sleep, their performance needs to be assessed against the current gold standard, i.e., polysomnography (PSG; measuring brain, eye, and muscle activity). Here, we assessed some commercially available sleep trackers, namely an activity tracker; Mi band (Xiaomi, Beijing, China), a scientific actigraph: Motionwatch 8 (CamNTech, Cambridge, UK), and a much-used mobile phone application: Sleep Cycle (Northcube, Gothenburg, Sweden). We recorded 27 nights in healthy sleepers using PSG and these devices and compared the results. Surprisingly, all devices had poor agreement with the PSG gold standard. Sleep parameter comparisons revealed that, specifically, the Mi band and the Sleep Cycle application had difficulties in detecting wake periods which negatively affected their total sleep time and sleep-efficiency estimations. However, all 3 devices were good in detecting the most basic parameter, the actual time in bed. In summary, our results suggest that, to date, the available sleep trackers do not provide meaningful sleep analysis but may be interesting for simply tracking time in bed. A much closer interaction with the scientific field seems necessary if reliable information shall be derived from such devices in the future.

Genomic alterations as mediators of miRNA dysregulation in ovarian cancer.

Ovarian cancer is the fifth most common cause of cancer death in women worldwide. Serous epithelial ovarian cancer (SEOC) is the most common and aggressive histological subtype. Widespread genomic alterations go hand-in-hand with aberrant DNA damage signaling and are a hallmark of high-grade SEOC. MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that are nonrandomly distributed in the genome. They are frequently located in chromosomal regions susceptible to copy number variation (CNV) associated with malignancy that can influence their expression. Widespread changes in miRNA expression have been reported in multiple cancer types including ovarian cancer. This review examines CNV and single nucleotide polymorphisms, two common types of genomic alterations that occur in ovarian cancer, in the context of their influence on the expression of miRNA and the ability of miRNA to bind to and regulate their target genes. This includes genes encoding proteins involved in DNA repair and the maintenance of genomic stability. Improved understanding of mechanisms of miRNA dysregulation and the role of miRNA in ovarian cancer will provide further insight into the pathogenesis and treatment of this disease.

Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer.

BACKGROUND: There is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC). MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers. METHODS: To identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 µL aliquots of serum collected from patients with SEOC (n = 28) and age-matched healthy donors (n = 28). Serum microRNA levels were assessed by quantitative RT-PCR following preamplification. RESULTS: microRNA (miR)-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48) were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years) for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P < 0.05; 0.05; 0.0005 respectively) and in combination, miR-200b + miR-200c normalized to serum volume and miR-103 was the best predictive classifier of SEOC (ROC-AUC = 0.784). This predictive model (miR-200b + miR-200c) was further confirmed by leave one out cross validation (AUC = 0.784). CONCLUSIONS: We identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

The tumor suppressor CDC73 interacts with the ring finger proteins RNF20 and RNF40 and is required for the maintenance of histone 2B monoubiquitination.

Monoubiquitination of histone H2B is a dynamic post-translational histone modification associated with transcriptional elongation and the DNA damage response. To date, dysregulation of histone monoubiquitination has not been linked to pathogenic mutations in genes encoding proteins, or co-factors, catalyzing this modification. The tumor suppressor cell division cycle 73 (CDC73) is mutated and/or down-regulated in parathyroid carcinoma, renal, breast, gastric and colorectal tumors, as well as in the germline of patients with the familial disorder-hyperparathyroidism jaw tumor syndrome. Using CDC73 as bait in a yeast two-hybrid assay, we identified the ring finger proteins RNF20 and RNF40 as binding partners of this tumor suppressor. These polypeptides constitute a heterodimeric complex that functions as the E3 ubiquitin ligase for monoubiquitination of histone H2B at lysine 120 (H2B-K120). We show that RNF20 and RNF40 bind to discrete, but closely located, residues on CDC73. Monoubiquitinated H2B-K120 was significantly reduced after loss of nuclear CDC73, both in vitro upon down-regulation of CDC73, and in CDC73 mutant parathyroid tumors. A second histone modification, trimethylation of histone 3 at lysine 4 (H3-K4me3), remained unchanged in the presence of mutant or down-regulated CDC73, suggesting that H3-K4me3 is not always tightly linked to H2B-K120 monoubiquitination for transcription as previously described. This is the first report of pathogenic mutations affecting histone monoubiquitination. We conclude that CDC73 is required for the maintenance of H2B-K120 monoubiquitination and propose that reduction in levels of monoubiquitinated H2B-K120 is a major mechanism whereby mutations in CDC73 exert their tumorigenic effect.

The chemokine CXCL1 induces proliferation in epithelial ovarian cancer cells by transactivation of the epidermal growth factor receptor.

The chemokine CXCL1 is elevated in plasma and ascites from patients with ovarian cancer. We have previously shown that CXCL1 is a marker of phosphatidylinositol 3-kinase signalling in epithelial ovarian cancer (EOC) cell lines, a pathway that is commonly activated in ovarian tumours. To investigate whether CXCL1 also has functional significance in ovarian cancer, this chemokine was either down-regulated using siRNAs or overexpressed by transfection of CXCL1 into the EOC cell lines SKOV3 and OVCAR-3 and proliferation assessed over 7 days. Overexpression of CXCL1 increased proliferation of ovarian cancer cells over 7 days, while down-regulation was inhibitory. Treatment of cells with recombinant CXCL1 induced epidermal growth factor receptor (EGFR) phosphorylation at Y1068, indicating crosstalk between the CXCL1 G-protein-coupled receptor CXCR2 and the EGFR. CXCL1-induced proliferation was also decreased by inhibition of EGFR kinase activity and was dependent on extracellular matrix metalloproteinase-mediated release of heparin-binding EGF (HB-EGF). Involvement of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signalling was also evident since inhibition of both Ras and MEK activity decreased CXCL1-induced proliferation. CXCL1-induced ERK1/2 phosphorylation was inhibited by the MEK1 inhibitor PD98059; however, EGFR phosphorylation was unaffected, indicating that CXCL1 activation of MAPK signalling is downstream of the EGFR. Taken together, these data show that CXCL1 functions through CXCR2 to transactivate the EGFR by proteolytic cleavage of HB-EGF, leading to activation of MAPK signalling and increased proliferation of EOC cells.

CDC73/HRPT2 CpG island hypermethylation and mutation of 5'-untranslated sequence are uncommon mechanisms of silencing parafibromin in parathyroid tumors.

The tumor suppressor HRPT2/CDC73 is mutated in constitutive DNA from patients with the familial disorder hyperparathyroidism-jaw tumor syndrome and in approximately 70% of all parathyroid carcinomas. In a number of HRPT2 mutant tumors however, expression of the encoded protein parafibromin is lost in the absence of a clear second event such as HRPT2 allelic loss or the presence of a second mutation in this tumor suppressor gene. We sought to determine whether hypermethylation of a 713 bp CpG island extending 648 nucleotides upstream of the HRPT2 translational start site and 65 nucleotides into exon 1 might be a mechanism contributing to the loss of expression of parafibromin in parathyroid tumors. Furthermore, we asked whether mutations might be present in the 5'-untranslated region (5'-UTR) of HRPT2. We investigated a pool of tissue from 3 normal parathyroid glands, as well as 15 individual parathyroid tumor samples including 6 tumors with known HRPT2 mutations, for hypermethylation of the HRPT2 CpG island. Methylation was not identified in any specimens despite complete loss of parafibromin expression in two parathyroid carcinomas with a single detectable HRPT2 mutation and retention of the wild-type HRPT2 allele. Furthermore, no mutations of a likely pathogenic nature were identified in the 5'-UTR of HRPT2. These data strongly suggest that alternative mechanisms such as mutation in HRPT2 intronic regions, additional epigenetic regulation such as histone modifications, or other regulatory inactivation mechanisms such as targeting by microRNAs may play a role in the loss of parafibromin expression.

The effect of disease-associated HRPT2 mutations on splicing.

Mutations in the tumour suppressor HRPT2 occur in patients with parathyroid carcinoma, kidney tumours and Hyperparathyroidism-Jaw Tumour syndrome. Disruption of exonic splicing through mutation of donor/acceptor splice sites or exonic splice enhancer (ESE) sites leads to loss of function of a number of major tumour suppressors including BRCA1, APC and MLH1. Given that the effect of HRPT2 mutations on splicing has not been widely studied, we used an in vitro splicing assay to determine whether 17 HRPT2 mutations located in hot-spot and other exons predicted to disrupt ESE consensus sites led to aberrant splicing. Using two independent web-based prediction programs, the majority of these mutations were predicted to disrupt ESE consensus sites; however, aberrant splicing of HRPT2 transcripts was not observed. Canonical donor or acceptor splice site mutations were also investigated using this splicing assay and transcripts assessed from tumour tissue. Splice site mutations were shown to lead to either exon skipping or retention of intronic sequences through the use of cryptic splice sites comprised of non-classical splicing signals. Aberrant splicing caused by disruption of ESE sites does not appear to have a major role in HRPT2-associated disease; however, premature truncation of parafibromin as the result of canonical donor or acceptor splice site mutations is associated with pathogenicity. Functional splicing assays must be undertaken in order to confirm web-based software predictions of the modification of putative ESE sites by disease-associated mutations.

Nucleolar localization of parafibromin is mediated by three nucleolar localization signals.

Parafibromin is a putative tumor suppressor encoded by HRPT2 and implicated in parathyroid tumorigenesis. We previously reported a functional bipartite nuclear localization signal (NLS) at residues 125-139. We now demonstrate that parafibromin exhibits nucleolar localization, mediated by three nucleolar localization signals (NoLS) at resides 76-92, 192-194 and 393-409. These NoLS represent clusters of basic amino acids arginine and lysine, similar to those found in other nucleolar proteins, as well as being characteristic of NLSs. While parafibromin's bipartite NLS is the primary determinant of nuclear localization, it does not mediate nucleolar localization. In contrast, the three identified NoLSs play only a minor role in nuclear localization, but are critical for the nucleolar localization of parafibromin.

Molecular diagnosis of primary hyperparathyroidism in familial cancer syndromes.

In the last few years, causative genes have been identified for most of the familial hyperparathyroidism conditions. Germline mutations in the tumour suppressors multiple endocrine neoplasia type 1 (MEN1) and hyperparathyroidism 2 (HRPT2) provide a molecular diagnosis of multiple endocrine neoplasia type 1 and hyperparathyroidism jaw tumour syndrome, respectively. Germline mutations in the proto-oncogene RET (rearranged during transfection) provide a molecular diagnosis of multiple endocrine neoplasia type 2. Germline mutations of both MEN1 and, less frequently HRPT2, have been found in familial isolated hyperparathyroidism. A molecular diagnosis can now be incorporated into the management of patients with these conditions, however, the ease of diagnostics and value of genetic information in the context of clinical screening and early surgical intervention varies between these disorders. This review focuses on familial hyperparathyroidism and its known causative genes in the setting of neoplastic syndromes, with particular discussion of recent developments in the molecular diagnosis of parathyroid carcinoma.

Identification of a functional bipartite nuclear localization signal in the tumor suppressor parafibromin.

Parafibromin is a putative tumor suppressor encoded by HRPT2, mutations in which have been implicated in the familial tumor syndrome hyperparathyroidism jaw tumor syndrome (HPT-JT), and sporadic parathyroid carcinoma. Recently, parafibromin has been shown to be an accessory factor for RNA polymerase II as part of the human Paf 1 complex, suggesting, as has been shown for its yeast homologue (Cdc 73), that it may have a role as an important regulator of transcription. Parafibromin has also been shown to interact with a histone methyltransferase complex that methylates histone H3 and to inhibit proliferation when overexpressed in mammalian cell lines. Despite these findings, the cellular localization of parafibromin has been controversial, with reports of both nuclear and nucleocytoplasmic localization. We have expressed wild-type and mutant parafibromin tagged with enhanced green fluorescent protein and have identified a functional bipartite nuclear localization signal (NLS) at residues 125-139 (nucleotides 373-417), KRAADEVLAEAKKPR, that is evolutionarily conserved and critical for the nuclear localization of parafibromin. We have also shown that the C-terminal arm of this bipartite NLS plays the primary role in nuclear localization. In support of these findings, specific HRPT2 mutations identified in HPT-JT or sporadic parathyroid carcinoma predicted to truncate parafibromin upstream of or within this NLS disrupt nuclear localization.

Phorbol ester-induced cell death in PC-12 cells overexpressing Bcl-2 is dependent on the time at which cells are treated.

The aim of the present study was to investigate the involvement of PKC in Bcl-2 protection against serum withdrawal-induced apoptosis in PC-12 cells. Human Bcl-2 was overexpressed in PC-12 cells and was found to totally inhibit serum withdrawal-induced apoptosis. 12-O-tetradecanoylphorbol-13-acetate (TPA) could induce cell death in PC-12 cells that overexpressed Bcl-2, implicating protein kinase C (PKC) in Bcl-2 protection. However, TPA-induced cell death did not involve caspase-3 activation or DNA degradation, suggesting that Bcl-2 was still inhibiting these processes and that TPA was mediating cell death either downstream of Bcl-2 or via independent signalling pathways. High cytosolic and particulate protein levels of PKC delta were correlated with TPA-induced cell death suggesting that PKC delta positively regulated this cell death. However, substantial down-regulation of PKC by prolonged exposure to TPA did not reduce the frequency of TPA-induced cell death, raising the possibility that PKC delta did not regulate cell death alone. Surprisingly, TPA-induced cell death was dependent on the time at which cells were treated, suggesting that cells were changing with time. Supporting this idea, the cytosolic and particulate protein levels of PKC delta and were found to change with time, and may account for the time-dependent manner in which TPA induced cell death. This is the first report to show that sensitivity to drug induced cell death in a cultured cell line changes with time. Experimental and theoretical evidence suggests that many cellular constituents exhibit temporal variations, oscillations or rhythms due to feedback regulation. We believe that investigation of these temporal changes, how they alter cell function with time and how they might be manipulated in single cells as well as across cellular populations is paramount in furthering our understanding of cellular behaviour.