Neurofilament light compared to neuron-specific enolase as a predictor of unfavourable outcome after out-of-hospital cardiac arrest.

AIM: We compared the prognostic abilities of neurofilament light (NfL) and neuron-specific enolase (NSE) in patients resuscitated from out-of-hospital cardiac arrest (OHCA) of various aetiologies. METHODS: We analysed frozen blood samples obtained at 24 and 48 hours from OHCA patients treated in 21 Finnish intensive care units in 2010 and 2011. We defined unfavourable outcome as Cerebral Performance Category (CPC) 3-5 at 12 months after OHCA. We evaluated the prognostic ability of the biomarkers by calculating the area under the receiver operating characteristic curves (AUROCs [95% confidence intervals]) and compared these with a bootstrap method. RESULTS: Out of 248 adult patients, 12-month outcome was unfavourable in 120 (48.4%). The median (interquartile range) NfL concentrations for patients with unfavourable and those with favourable outcome, respectively, were 689 (146-1804) pg/mL vs. 31 (17-61) pg/mL at 24 h and 1162 (147-4360) pg/mL vs. 36 (21-87) pg/mL at 48 h, p < 0.001 for both. The corresponding NSE concentrations were 13.3 (7.2-27.3) µg/L vs. 8.5 (5.8-13.2) µg/L at 24 h and 20.4 (8.1-56.6) µg/L vs. 8.2 (5.9-12.1) µg/L at 48 h, p < 0.001 for both. The AUROCs to predict an unfavourable outcome were 0.90 (0.86-0.94) for NfL vs. 0.65 (0.58-0.72) for NSE at 24 h, p < 0.001 and 0.88 (0.83-0.93) for NfL and 0.73 (0.66-0.81) for NSE at 48 h, p < 0.001. CONCLUSION: Compared to NSE, NfL demonstrated superior accuracy in predicting long-term unfavourable outcome after OHCA.

European Academy of Neurology guideline on the diagnosis of coma and other disorders of consciousness.

BACKGROUND AND PURPOSE: Patients with acquired brain injury and acute or prolonged disorders of consciousness (DoC) are challenging. Evidence to support diagnostic decisions on coma and other DoC is limited but accumulating. This guideline provides the state-of-the-art evidence regarding the diagnosis of DoC, summarizing data from bedside examination techniques, functional neuroimaging and electroencephalography (EEG). METHODS: Sixteen members of the European Academy of Neurology (EAN) Scientific Panel on Coma and Chronic Disorders of Consciousness, representing 10 European countries, reviewed the scientific evidence for the evaluation of coma and other DoC using standard bibliographic measures. Recommendations followed the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. The guideline was endorsed by the EAN. RESULTS: Besides a comprehensive neurological examination, the following suggestions are made: probe for voluntary eye movements using a mirror; repeat clinical assessments in the subacute and chronic setting, using the Coma Recovery Scale - Revised; use the Full Outline of Unresponsiveness score instead of the Glasgow Coma Scale in the acute setting; obtain clinical standard EEG; search for sleep patterns on EEG, particularly rapid eye movement sleep and slow-wave sleep; and, whenever feasible, consider positron emission tomography, resting state functional magnetic resonance imaging (fMRI), active fMRI or EEG paradigms and quantitative analysis of high-density EEG to complement behavioral assessment in patients without command following at the bedside. CONCLUSIONS: Standardized clinical evaluation, EEG-based techniques and functional neuroimaging should be integrated for multimodal evaluation of patients with DoC. The state of consciousness should be classified according to the highest level revealed by any of these three approaches.

Rivaroxaban versus aspirin for secondary prevention of ischaemic stroke in patients with cancer: a subgroup analysis of the NAVIGATE ESUS randomized trial.

BACKGROUND AND PURPOSE: Cancer is a frequent finding in ischaemic stroke patients. The frequency of cancer amongst participants in the NAVIGATE ESUS randomized trial and the distribution of outcome events during treatment with aspirin and rivaroxaban were investigated. METHODS: Trial participation required a recent embolic stroke of undetermined source. Patients' history of cancer was recorded at the time of study entry. During a mean follow-up of 11 months, the effects of aspirin and rivaroxaban treatment on recurrent ischaemic stroke, major bleeding and all-cause mortality were compared between patients with cancer and patients without cancer. RESULTS: Amongst 7213 randomized patients, 543 (7.5%) had cancer. Of all patients, 3609 were randomized to rivaroxaban [254 (7.0%) with cancer] and 3604 patients to aspirin [289 (8.0%) with cancer]. The annual rate of recurrent ischaemic stroke was 4.5% in non-cancer patients in the rivaroxaban arm and 4.6% in the aspirin arm [hazard ratio (HR) 0.98, 95% confidence interval (CI) 0.78-1.24]. In cancer patients, the rate of recurrent ischaemic stroke was 7.7% in the rivaroxaban arm and 5.4% in the aspirin arm (HR 1.43, 95% CI 0.71-2.87). Amongst cancer patients, the annual rate of major bleeds was non-significantly higher for rivaroxaban than aspirin (2.9% vs. 1.1%; HR 2.57, 95% CI 0.67-9.96; P for interaction 0.95). All-cause mortality was similar in both groups. CONCLUSIONS: Our exploratory analyses show that patients with embolic stroke of undetermined source and a history of cancer had similar rates of recurrent ischaemic strokes and all-cause mortality during aspirin and rivaroxaban treatments and that aspirin appeared safer than rivaroxaban in cancer patients regarding major bleeds. www.clinicaltrials.gov (NCT02313909).

Sharp and round shapes of seen objects have distinct influences on vowel and consonant articulation.

The shape and size-related sound symbolism phenomena assume that, for example, the vowel [i] and the consonant [t] are associated with sharp-shaped and small-sized objects, whereas [ɑ] and [m] are associated with round and large objects. It has been proposed that these phenomena are mostly based on the involvement of articulatory processes in representing shape and size properties of objects. For example, [i] might be associated with sharp and small objects, because it is produced by a specific front-close shape of articulators. Nevertheless, very little work has examined whether these object properties indeed have impact on speech sound vocalization. In the present study, the participants were presented with a sharp- or round-shaped object in a small or large size. They were required to pronounce one out of two meaningless speech units (e.g., [i] or [ɑ]) according to the size or shape of the object. We investigated how a task-irrelevant object property (e.g., the shape when responses are made according to size) influences reaction times, accuracy, intensity, fundamental frequency, and formant 1 and formant 2 of vocalizations. The size did not influence vocal responses but shape did. Specifically, the vowel [i] and consonant [t] were vocalized relatively rapidly when the object was sharp-shaped, whereas [u] and [m] were vocalized relatively rapidly when the object was round-shaped. The study supports the view that the shape-related sound symbolism phenomena might reflect mapping of the perceived shape with the corresponding articulatory gestures.

Symptomatic post-thrombolytic intracerebral hemorrhage is not related to the cause of stroke.

BACKGROUND AND PURPOSE: The development of intracerebral hemorrhage following intravenous thrombolysis (IVT) can be influenced by various confounders related to the underlying vessel and tissue conditions. There are some data on association of cause of the stroke and the hemorrhage transformation. We tested the hypothesis that the cause of stroke is associated with the development of symptomatic intracerebral hemorrhage (sICH) following IVT. METHODS: A consecutive cohort of 2485 IVT-treated patients at the Helsinki University Central Hospital was classified according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) criteria. An sICH was classified according to the European Cooperative Acute Stroke Study II criteria. The associations of sICH with nominal, ordinal and continuous variables were analyzed in a univariate binary regression model and adjusted in multivariate binary regression models. RESULTS: In univariate analyses, cardioembolism [odds ratio (OR), 1.14; 95% confidence interval (CI), 0.79-1.64] and large-artery atherosclerosis (OR, 1.30; 95% CI, 0.85-2.00) were not associated with sICH, and small-vessel occlusion was associated with lower odds for sICH (OR, 0.18; 95% CI, 0.06-0.57). When adjusted for previously identified factors associated with sICH, none of the TOAST categories was associated with a higher or lower frequency of sICH. CONCLUSIONS: The development of sICH in IVT-treated patients is not related to the cause of stroke.

Recognition of posterior circulation stroke.

OBJECTIVES: Better identification and triage of acute posterior circulation (PC) stroke patients is needed as the PC ischemic stroke (IS) patients may be allowed longer thrombolysis window than anterior circulation (AC) IS patients and PC patients with hemorrhagic stroke (ICH) may require care in a neurosurgical unit possibly remote from stroke unit. MATERIALS AND METHODS: Consecutive stroke patients treated at a tertiary center with thrombolysis (100% for IS) and/or comprehensive stroke unit care. RESULTS: Altogether, 1641 patients had AC (75%) and 553 PC strokes. The PC-IS patients were younger (65 vs 70), had less often prior hypertension (51 vs 61%), and were twice more often on warfarin. They presented 3.5 times more often with seizure, vomited five times more often, had headache twice as often, and required intubation 2 to 3 times more often despite equal NIHSS (9 vs 8) or GCS (15 both) scores with AC-IS patients. Among PC patients, IS (n = 190) associated with younger age, prior atrial fibrillation (AF) in 25% and dyslipidemia in ~40%. One-third of PC-ICH patients (n = 363) had headache and vomited at the onset. PC-ICH patients had BP median of 177/92 mmHg and blood glucose 7.4 mmol/l on ER arrival. Warfarin use was twice as common in PC-ICH. CONCLUSIONS: Despite being of typical age for multiple cardiovascular conditions the PC-ICH patients less often have a previous history of AF or dyslipidemia than IS patients do. The vomiting PC-ICH patient with hypertensive BP values often has headache and a red flag for hemorrhage is warfarin treatment.

Shared processing of planning articulatory gestures and grasping.

It has been proposed that articulatory gestures are shaped by tight integration in planning mouth and hand acts. This hypothesis is supported by recent behavioral evidence showing that response selection between the precision and power grip is systematically influenced by simultaneous articulation of a syllable. For example, precision grip responses are performed relatively fast when the syllable articulation employs the tongue tip (e.g., [te]), whereas power grip responses are performed relatively fast when the syllable articulation employs the tongue body (e.g., [ke]). However, this correspondence effect, and other similar effects that demonstrate the interplay between grasping and articulatory gestures, has been found when the grasping is performed during overt articulation. The present study demonstrates that merely reading the syllables silently (Experiment 1) or hearing them (Experiment 2) results in a similar correspondence effect. The results suggest that the correspondence effect is based on integration in planning articulatory gestures and grasping rather than requiring an overt articulation of the syllables. We propose that this effect reflects partially overlapped planning of goal shapes of the two distal effectors: a vocal tract shape for articulation and a hand shape for grasping. In addition, the paper shows a pitch-grip correspondence effect in which the precision grip is associated with a high-pitched vocalization of the auditory stimuli and the power grip is associated with a low-pitched vocalization. The underlying mechanisms of this phenomenon are discussed in relation to the articulation-grip correspondence.

Phase II trial of PTK787/ZK 222584 (vatalanib) administered orally once-daily or in two divided daily doses as second-line monotherapy in relapsed or progressing patients with stage IIIB/IV non-small-cell lung cancer (NSCLC).

BACKGROUND: The objective of this multicenter, prospective uncontrolled phase II trial was to determine efficacy, safety and tolerability of vatalanib, an oral angiogenesis inhibitor targeting all known vascular endothelial growth factor receptors, in the second-line treatment of non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with stage IIIB/IV NSCLC-proven tumor progression during or after one platinum-based chemotherapy regimen received a fixed dose of 1250 mg vatalanib either once-daily dosing (QD) or two divided daily dosing (TDD: 500 mg a.m. + 750 mg p.m.) until disease progression or unacceptable toxicity. Primary end point was the disease control rate (DCR) at 12 weeks. RESULTS: Fifty-four and 58 patients were enrolled to the QD and TDD arms. DCR at 12 weeks was 35% in the QD and 37% in the TDD arm. The best overall response included one (2%) patient with confirmed partial response with QD and three (5%) with TDD. Median progression-free survival and overall survival were 2.1/7.3 months in the QD arm and 2.8/9.0 months with TDD arm. This therapy showed a moderate toxicity profile for the majority of patients. CONCLUSIONS: In the chosen patient population, vatalanib QD and TDD dosing demonstrated potential benefits in tumor size reduction, DCR, and survival.

The functional status and perceived quality of life in long-term survivors of out-of-hospital cardiac arrest.

BACKGROUND: Limited data exist on how long-term survivors after pre-hospital cardiac arrest lead their lives. This study assessed functional status and perceived quality of life in patients surviving for 15 years after successful resuscitation from witnessed out-of-hospital cardiac arrest as a result of ventricular fibrillation. METHODS: A 15-year follow-up study of 59 1-year survivors after successful pre-hospital resuscitation who were thoroughly evaluated at 3 and 12 months after out-of-hospital cardiac arrest. Eleven patients were still alive 15 years later. Ten of them were reached and underwent a comprehensive neuropsychological and neurological examination. Cognitive performance was evaluated and compared with individual results 15 years earlier and with an age-matched control group. The cause and time of death of the non-survivors were established. RESULTS: All 10 evaluated long-term survivors lived at home and were independent in their activities of daily living. Their mean age was 72 years. In nine patients there was no change in the present neurological status compared with the status at 1 year after resuscitation, and in one patient it had improved. Five patients were cognitively intact. In four patients mild cognitive problems had emerged or slightly progressed. All but one were satisfied with their perceived quality of life. By the time of examination, the mean survival time for the 1-year survivors was 7 years, and the mean age at the time of death was 70 years. CONCLUSION: Once good outcome after cardiac arrest is achieved, it can be maintained for more than 10 years.

Therapeutic hypothermia after prolonged cardiac arrest due to non-coronary causes.

Mild resuscitative hypothermia has been shown to improve neurological outcome after cardiac arrest presenting with ventricular fibrillation (VF) due to cardiac causes. We describe the experience of inducing mild hypothermia in three patients with non-cardiac causes of arrest and long delays before a return of spontaneous circulation (ROSC). In one patient, extreme metabolic acidosis due to inadvertent oesophageal intubation complicated therapy, and the role of point-of-care diagnostics in the prehospital setting is briefly discussed. All patients survived to discharge from hospital, and neuropsychological examinations revealed good recovery. It is concluded that mild resuscitative hypothermia may be beneficial also in patients with obvious non-coronary causes for cardiac arrest.

Reconstitution of cyclin D1-associated kinase activity drives terminally differentiated cells into the cell cycle.

Terminal cell differentiation entails definitive withdrawal from the cell cycle. Although most of the cells of an adult mammal are terminally differentiated, the molecular mechanisms preserving the postmitotic state are insufficiently understood. Terminally differentiated skeletal muscle cells, or myotubes, are a prototypic terminally differentiated system. We previously identified a mid-G(1) block preventing myotubes from progressing beyond this point in the cell cycle. In this work, we set out to define the molecular basis of such a block. It is shown here that overexpression of highly active cyclin E and cdk2 in myotubes induces phosphorylation of pRb but cannot reactivate DNA synthesis, underscoring the tightness of cell cycle control in postmitotic cells. In contrast, forced expression of cyclin D1 and wild-type or dominant-negative cdk4 in myotubes restores physiological levels of cdk4 kinase activity, allowing progression through the cell cycle. Such reactivation occurs in myotubes derived from primary, as well as established, C2C12 myoblasts and is accompanied by impairment of muscle-specific gene expression. Other terminally differentiated systems as diverse as adipocytes and nerve cells are similarly reactivated. Thus, the present results indicate that the suppression of cyclin D1-associated kinase activity is of crucial importance for the maintenance of the postmitotic state in widely divergent terminally differentiated cell types.

Specificity of Cdk activation in vivo by the two Caks Mcs6 and Csk1 in fission yeast.

Activating phosphorylation of cyclin-dependent kinases (Cdks) is mediated by at least two structurally distinct types of Cdk-activating kinases (Caks): the trimeric Cdk7-cyclin H-Mat1 complex in metazoans and the single-subunit Cak1 in budding yeast. Fission yeast has both Cak types: Mcs6 is a Cdk7 ortholog and Csk1 a single-subunit kinase. Both phosphorylate Cdks in vitro and rescue a thermosensitive budding yeast CAK1 strain. However, this apparent redundancy is not observed in fission yeast in vivo. We have identified mutants that exhibit phenotypes attributable to defects in either Mcs6-activating phosphorylation or in Cdc2-activating phosphorylation. Mcs6, human Cdk7 and budding yeast Cak1 were all active as Caks for Cdc2 when expressed in fission yeast. Although Csk1 could activate Mcs6, it was unable to activate Cdc2. Biochemical experiments supported these genetic results: budding yeast Cak1 could bind and phosphorylate Cdc2 from fission yeast lysates, whereas fission yeast Csk1 could not. These results indicate that Mcs6 is the direct activator of Cdc2, and Csk1 only activates Mcs6. This demonstrates in vivo specificity in Cdk activation by Caks.

Kaposi's sarcoma-associated herpesvirus-encoded v-cyclin triggers apoptosis in cells with high levels of cyclin-dependent kinase 6.

Kaposi's sarcoma-associated herpesvirus (KSHV) has a key etiological role in development of Kaposi's sarcoma (KS). v-Cyclin is a KSHV-encoded homologue to D-type cyclins that associates with cellular cyclin-dependent kinase 6 (CDK6). v-Cyclin promotes S-phase entry of quiescent cells and has been suggested to execute functions of both D- and E-type cyclins. In this study, expression of v-cyclin in cells with elevated levels of CDK6 led to apoptotic cell death after the cells entered S phase. The cell death required the kinase activity of CDK6 because cells expressing a kinase-deficient form of CDK6 did not undergo apoptosis upon v-cyclin expression. Studies on the mechanisms involved in this caspase-3-mediated apoptosis indicated that it was independent of cellular p53 or pRb status, and it was not suppressed by Bcl-2. In contrast, the KSHV-encoded v-Bcl-2 efficiently suppressed v-cyclin-/CDK6-induced apoptosis, demonstrating a marked difference in the antiapoptotic properties of c-Bcl-2 and v-Bcl-2. In KS lesions, high CDK6 expression was confined to a subset of cells, some of which displayed signs of apoptosis. These results suggest that v-cyclin may exert both growth-promoting and apoptotic functions in KS, depending on factors regulating CDK6 and v-Bcl-2 levels.

E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells.

We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here that overexpression of E2F-1, E2F-2 and E2F-4, or a chimeric E2F-4 tethered to a nuclear localization signal cannot reactivate postmitotic skeletal muscle cells (myotubes). This is not due to lack of transcriptional activity, as demonstrated on both a reporter construct and a number of endogenous target genes. Although cyclin E was strongly overexpressed in E2F-transduced myotubes, it lacked associated kinase activity, possibly explaining the inability of the myotubes to enter S phase and accumulate cyclin A. Although E2F is not sufficient to trigger DNA synthesis in myotubes, its activity is necessary even in the presence of E1A, as dominant-negative DP-1 mutants inhibit E1A-mediated cell cycle reentry. Our data show that, to reactivate myotubes, E1A must exert other functions, in addition to releasing E2F. They also establish mouse myotubes as an experimental system uniquely suited to study the most direct E2F functions in the absence of downstream cell cycle effects.

Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest.

Germ-line mutations of LKB1 (STK11) lead to Peutz-Jeghers syndrome characterized by gastrointestinal polyps and cancer of different organ systems. The mutations lead to loss or severe impairment of Lkb1 serine/threonine kinase activity. Therefore LKB1 has been implicated as a tumor suppressor gene, but only a few mutations in the coding exons of LKB1 have been detected in sporadic tumors. Here, we have identified tumor cell lines with severely reduced mRNA levels and impaired Lkb1 kinase activity. Reintroducing Lkb1 into these cells suppressed cell growth. The Lkb1-mediated growth inhibition was caused by a G(1) cell cycle block and was not detected with several naturally occurring Lkb1 mutants. These results indicate that LKB1 has functional and specific growth-suppressing activity.

Expression of LKB1 and PTEN tumor suppressor genes during mouse embryonic development.

Germ-line mutations of LKB1 and PTEN tumor suppressor genes underlie the phenotypically related Peutz-Jeghers syndrome (PJS) and Cowden disease (CD), respectively. To analyze possible developmental roles of PTEN and LKB1, we have studied their mRNA expression during mouse embryonic development (E7-17.5) by in situ hybridization. Ubiquitous expression of both genes during early stages (E7-11) became more restricted in later embryonic development (E15-19) where LKB1 and PTEN showed prominent overlapping expression in e.g. gastrointestinal tract and lung. In contrast, LKB1 was selectively expressed at high levels in testis and PTEN was prominently expressed in skin epithelium and underlying mesenchyme. These results indicate that LKB1 and PTEN display largely overlapping expression patterns during embryonic development. Moreover, a high expression of these genes was observed in the tissues and organs affected in PJS and CD patients and in PTEN+/- mice.

Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex.

The observation that cyclin B1 protein and mRNAs are down-regulated in terminally differentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-off of cyclin B1 promoter activity in differentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation.

Mutations and impaired function of LKB1 in familial and non-familial Peutz-Jeghers syndrome and a sporadic testicular cancer.

Germline mutations in LKB1 have been reported to underlie familial Peutz-Jeghers syndrome (PJS) with intestinal hamartomatous polyps and an elevated risk of various neoplasms. To investigate the prevalence of LKB1 germline mutations in PJS more generally, we studied samples from 33 unrelated PJS patients including eight non-familial sporadic patients, 20 familial patients and five patients with unknown family history. Nineteen germline mutations were identified, 12 (60%) in familial and four (50%) in sporadic cases. LKB1 mutations were not detected in 14 (42%) patients, indicating that the existence of additional minor PJS loci cannot be excluded. LKB1 is predicted to encode a serine/threonine kinase. To demonstrate the putative Lkb1 kinase function and to study the consequences of LKB1 mutations in PJS and sporadic tumors, we have analyzed the kinase activity of wild-type and mutant Lkb1 proteins. Interestingly, while most of the small deletions or missense mutations resulted in loss-of-function alleles, one missense mutation (G163D) previously identified in a sporadic testicular tumor demonstrated severely impaired but detectable kinase activity.

Expression of E1A in terminally differentiated muscle cells reactivates the cell cycle and suppresses tissue-specific genes by separable mechanisms.

Terminally differentiated cells are characterized by permanent withdrawal from the cell cycle; they do not enter S phase even when stimulated by growth factors or retroviral oncogenes. We have shown, however, that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. In this report, we describe the molecular events triggered by E1A in terminally differentiated skeletal muscle cells. We found that in myotubes infected with the adenovirus mutant dl520, 12S E1A bypasses the early G1 phase and activates the expression of late-G1 genes, such as the cyclin E and cyclin A genes, cdk2, PCNA, and B-myb. Of these, the cyclin E gene and cdk2 were significantly overexpressed in comparison with levels in proliferating, undifferentiated myoblasts. p130 and pRb were phosphorylated before the infected myotubes entered S phase, despite the high expression of the cyclin-dependent kinase inhibitor p21, and E2F was released. Our results suggest that one of the mechanisms that E1A uses to overcome the proliferative block of terminally differentiated cells involves coordinated overexpression of cyclin E and cdk2. Following E1A expression, the myogenic transcription factors MyoD and myogenin and the muscle-specific structural genes encoding muscle creatine kinase and myosin heavy chain were downregulated. The muscle regulatory factors were also silenced in myotubes infected with adenovirus E1A mutants incapable of reactivating the cell cycle in terminally differentiated muscle cells. Thus, the suppression of the differentiation program is not a consequence of cell cycle reactivation in myotubes, and it is induced by an independent mechanism. Our results show that E1A reactivates the cell cycle and suppresses tissue-specific gene expression in terminally differentiated muscle cells, thus causing dedifferentiation.

Terminally differentiated skeletal myotubes are not confined to G0 but can enter G1 upon growth factor stimulation.

Terminally differentiated cells are specialized cells unable to proliferate that constitute most of the mammalian body. Despite their abundance, little information exists on the characteristics of cell cycle control in these cells and the molecular mechanisms that prevent their proliferation. They are generally believed to be irreversibly restricted to the G0 state. In this report, we define some features of a paradigmatic terminally differentiated system, the skeletal muscle, by studying its responses to various mitogenic stimuli. We show that forced expression of a number of cell cycle-regulatory genes, including erbB-2, v-ras, v-myc, B-myb, ld-1, and E2F-1, alone or in combinations, cannot induce terminally differentiated skeletal muscle cells (myotubes) to synthesize DNA. However, serum-stimulated myotubes display a typical immediate-early response, including the up-regulation of c-fos, c-jun, c-myc, and ld-1. They also elevate the expression of cyclin D1 after 4 hours of serum treatment. All these events take place in myotubes in a way that is indistinguishable from that of quiescent, undifferentiated myoblasts reactivated by serum. Moreover, pretreatment with serum shortens the time required by E1A to induce DNA synthesis, confirming that myotubes can partially traverse G1. Serum growth factors do not activate late-G1 genes in myotubes, suggesting that the block that prevents terminally differentiated cells from proliferating acts in mid-G1. Our results show that terminally differentiated cells are not confined to G0 but can partially reenter G1 in response to growth factors; they contribute to a much-needed definition of terminal differentiation. The important differences in the control of the cell cycle between terminally differentiated and senescent cells are discussed.