The cell cycle inhibitor p27 Kip1 also has cyclin-cyclin-dependent kinase (CDK)-independent functions. To investigate the significance of these functions in vivo, we generated a knock-in mouse in which four amino acid substitutions in the cdkn1b gene product prevent its interaction with cyclins and CDKs (p27 CK − ). In striking contrast to complete deletion of the cdkn1b gene, which causes spontaneous tumorigenesis only in the pituitary, the p27 CK − protein dominantly caused hyperplastic lesions and tumors in multiple organs, including the lung, retina, pituitary, ovary, adrenals, spleen, and lymphomas. Moreover, the high incidence of spontaneous tumors in the lung and retina was associated with amplification of stem/progenitor cell populations. Therefore, independently of its role as a CDK inhibitor, p27 Kip1 promoted stem cell expansion and functioned as a dominant oncogene in vivo. Thus, the p27 CK − mouse unveils a dual role for p27 during tumorigenesis: It is a tumor suppressor by virtue of its cyclin-CDK regulatory function, and also an oncogene through a cyclin-CDK-independent function. This may explain why the cdkn1b gene is rarely inactivated in human tumors, and the p27 CK − mouse in which the tumor suppressor function is lost but the cyclin-CDK-independent-oncogenic-function is maintained may represent a more faithful model for the widespread role of p27 misregulation in human cancers than the p27 null.[Keywords: p27 Kip1 ; lung tumor; oncogene; retina; bronchioalveolar stem cell; desquamative interstitial pneumonitis] Supplemental material is available at http://www.genesdev.org.

It was previously reported that the ciliary epithelium (CE) of the mammalian eye contains a rare population of cells that could produce clonogenic self-renewing pigmented spheres in culture. Based on their ability to up-regulate genes found in retinal neurons, it was concluded that these sphere-forming cells were retinal stem cells. This conclusion raised the possibility that CE-derived retinal stem cells could help to restore vision in the millions of people worldwide who suffer from blindness associated with retinal degeneration. We report here that human and mouse CE-derived spheres are made up of proliferating pigmented ciliary epithelial cells rather than retinal stem cells. All of the cells in the CE-derived spheres, including the proliferating cells, had molecular, cellular, and morphological features of differentiated pigmented CE cells. These differentiated cells ectopically expressed nestin when exposed to growth factors and low levels of pan-neuronal markers such as beta-III-tubulin. Although the cells aberrantly expressed neuronal markers, they retained their pigmented CE cell morphology and failed to differentiate into retinal neurons in vitro or in vivo. Our results provide an example of a differentiated cell type that can form clonogenic spheres in culture, self-renew, express progenitor cell markers, and initiate neuronal differentiation that is not a stem or progenitor cell. More importantly, our findings highlight the importance of shifting the focus away from studies on CE-derived spheres for cell-based therapies to restore vision in the degenerating retina and improving techniques for using ES cells or retinal precursor cells.O ver 40 million people worldwide are blind. Macular degeneration accounts for Ϸ8 million cases of blindness. Although the cause of macular degeneration is not known, 1 potential treatment is cell therapy. Stem cells hold great promise for regenerative medicine, and recently many efforts have been devoted to finding suitable candidates for retinal transplants. These candidates include photoreceptor progenitors (1) and embryonic stem cells (2, 3). Others have looked to the ciliary epithelium (CE) as a potential source of retinal stem cells (4, 5).In 2000, Tropepe and colleagues revealed that the CE of the mouse eye can be dissociated, maintained in culture at clonal density and stimulated to form clonogenic spheres (5). The CEderived spheres were pigmented, expressed nestin, and could be dissociated and replated to form spheres up to 8 times. When transferred to differentiation conditions in culture, cells from the CE-derived spheres up-regulated genes found in rod photoreceptors, bipolar neurons, and Müller glia (5). These findings were later extended to CE isolated from postmortem human eyes (4). Based on these and other data, it was suggested that the sphere-forming cells in the mammalian CE are retinal stem cells (RSCs) and, thus, hold promise for therapeutic replacement of retinal neurons lost to disease or degeneration.Given the potential impact of cell replacement ...

During neurogenesis, the progression from a progenitor cell to a differentiated neuron is believed to be unidirectional and irreversible. The Rb family of proteins (Rb, p107, and p130) regulates cell-cycle exit and differentiation during retinogenesis. Rb and p130 are redundantly expressed in the neurons of the inner nuclear layer (INL) of the retina. We have found that in the adult Rb;p130-deficient retinae p107 compensation prevents ectopic proliferation of INL neurons. However, p107 is haploinsufficient in this process. Differentiated Rb(-/-);p107(+/-);p130(-/-) horizontal interneurons re-entered the cell cycle, clonally expanded, and formed metastatic retinoblastoma. Horizontal cells were not affected in Rb(+/-);p107(-/-);p130(-/-) or Rb(-/-);p107(-/-);p130(+/-), retinae suggesting that one copy of Rb or p130 was sufficient to prevent horizontal proliferation. We hereby report that differentiated neurons can proliferate and form cancer while maintaining their differentiated state including neurites and synaptic connections.

The p53 pathway is disrupted in virtually every human tumor. In ϳ50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. Tumorigenesis is a multistep process that involves dysregulation of several pathways that are crucial for cell growth and survival (1). The p53 pathway regulates cell survival in response to cellular stress (e.g. DNA damage) or oncogenic stress (e.g. Rb pathway dysregulation) (2, 3) and is suppressed in virtually every human cancer by genetic lesions in the p53 gene or other components of the pathway (4). Approximately half of all cancers express wild-type p53, and considerable research over the past decade has focused on inducing p53-mediated cell death in these tumors (4, 5). Most efforts to date have focused on inhibiting MDM2, a negative regulator of p53 (6 -14).Another key regulator of the p53 pathway is a protein related to MDM2 called MDMX (15-17). MDM2 and MDMX share homology in their p53-binding domains, but MDMX is believed to regulate p53 through distinct mechanisms. Specifically, MDM2 primarily regulates p53 stability and subcellular localization, whereas MDMX may directly regulate p53 transcription (17-21). MDMX is genetically amplified in 19% of breast carcinomas, 19% of colon carcinomas, 18% of lung carcinomas, and a smaller percentage of gliomas (17). One of the best characterized tumors with an MDMX amplification is retinoblastoma. Approximately 65% of human retinoblastomas have increased MDMX copy number, which correlates with increased MDMX mRNA and protein (22). Previous studies have demonstrated that the MDMX amplification suppresses p53-mediated cell death in Rb pathway-deficient retinoblasts (22).A general consensus is emerging that to efficiently induce a p53 response in tumor cells that express wild-type p53, it may be necessary to inactivate both MDM2 and MDMX (18,23,24). To date, no screens to identify small molecule inhibitors of MDMX have been reported, and MDM2 inhibitors probably do not bind as efficiently to MDMX because of structural differences in the p53-binding pockets of the two proteins (25-27). Consistent with this theory, nutlin-3a binds MDMX with at least a 40-fold weaker equilibrium binding constant than for MDM2 (22). Therefore, to identify small molecules that bind MDMX and prevent its interaction with p53, we developed biochemical and cell-based assays suitable for high throughput screening (HTS)

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase with significant homology to cell cycle-related Cdks but is not believed to be active in a typical cell cycle. In Cdk5-deficient embryos and Cdk5 chimeras, migration and survival of postmitotic neurons is compromised in a cell-autonomous manner. In the present study, we show that loss of Cdk5 leads to both failure of neuronal differentiation and loss of cell cycle control. Using specific cytoskeletal proteins as indices of neuronal differentiation, we find that Cdk5-deficient neurons are significantly arrested or delayed in their developmental program both in vivo and in vitro.

Cyclin-dependent kinase 5 (Cdk5) is a nontraditional Cdk that is primarily active in postmitotic neurons. Its best known substrates are cytoskeletal proteins. Less appreciated is its role in the maintenance of a postmitotic state. We show here that in cycling cells (NIH 3T3), the localization of Cdk5 changes from predominantly nuclear to cytoplasmic as cells reenter a cell cycle after serum starvation. Similarly, when ␤-amyloid peptide is used to stimulate cultured primary neurons to reenter a cell cycle, they too show a loss of nuclear Cdk5. Blocking nuclear export pharmacologically abolishes cell cycle reentry in wild-type but not Cdk5 ؊/؊ neurons, suggesting a Cdk5-specific effect. Cdk5 overexpression targeted to the nucleus of Cdk5 ؊/؊ neurons effectively blocks the cell cycle, but cytoplasmic targeting is ineffective. Further, in both human Alzheimer's disease as well as in the R1.40 mouse Alzheimer's model and the E2f1 ؊/؊ mouse, neurons expressing cell cycle markers consistently show reduced nuclear Cdk5. Thus, both in vivo and in vitro, neurons that reenter a cell cycle lose nuclear Cdk5. We propose that the nuclear Cdk5 plays an active role in allowing neurons to remain postmitotic as they mature and that loss of nuclear Cdk5 leads to cell cycle entry.Alzheimer's disease ͉ ␤-amyloid precursor protein transgenic mice ͉ cell cycle ͉ E2F1 ͉ neurodegeneration

Alzheimer's disease is late life dementia associated with significant neurodegeneration in both cortical and subcortical regions. During the ϳ10 year course of the disease, neurons are lost in a progressive pattern that is relatively consistent among individuals. One example of this is the progression of disease pathology found in both the neocortex and archicortex. In these structures, the earliest problems can be found in superficial cortical layers (II-IV), whereas later the disease advances to involve the deeper cortical layers (V-VI). It is unclear whether these apparent differences in sensitivity are intrinsic to the neurons or imposed by external factors such as the pattern of connections. We used ␤-amyloid (A␤) peptide treatment of cultured mouse neurons as our model system. We show first that, as in hippocampus, dissociated cultures of embryonic cortical neurons are biased toward the survival of cells that were finishing division in the ventricular zone at the time of harvest. Thus, embryonic day 13.5 (E13.5) cultures contain primarily deep-layer neurons whereas E16.5 cultures contain cells destined for upper layers. We use this cell-type specific segregation to our advantage and show, using both differences in gene expression profiles and A␤ survival curves, that deeper layer neurons are significantly more resistant to the toxic effects of A␤ than are cells from the more superficial strata. This suggests that an intrinsic underlying biology drives at least part of the AD progression pattern and that the time of harvest is a crucial variable in the interpretation of any cortical culture experiment.

Tardive dyskinesia (TD) is a drug-induced movement disorder (DIMD) seen in patients taking dopamine-receptor blocking agents (DRBAs). Clinicians should regularly monitor patients with or at risk of developing DIMDs; however, telehealth visits during the COVID-19 pandemic presented several significant challenges related to screening and care of these patients. In this observational survey study, respondents compared in-person with video/telephone visits to determine the impact on the evaluation, diagnosis, and monitoring of patients with DIMDs. Methods: The online survey was conducted (May 14-June 21, 2021) with qualified clinicians who prescribed a vesicular monoamine transporter 2 inhibitor or benztropine for DIMDs in the past 6 months, spent ≤70% of their professional time in the clinic, and conducted telehealth visits with ≥15% of their patients between December 2020 and January 2021. The questionnaire probed clinicians about their ability to evaluate, diagnose and monitor (hereinafter referred to as manage) patients with DIMDs via telehealth. Results: Survey respondents included 277 clinicians from psychiatry (n = 168) and neurology (n = 109) practices. Certain signs and symptoms (visual cues) used for diagnosis of DIMDs were not observable through telehealth and evaluation was comparatively more difficult with phone visits than video visits. Patients without caregivers and lower-functioning patients were at higher risk of missed diagnosis of DIMDs and were also difficult to monitor via telehealth. Limited access to computers or telephones and patients living alone were among the top socioeconomic barriers limiting clinicians' ability to diagnose DIMDs. Patients without a regular caregiver were also more difficult for clinicians to evaluate and monitor adequately. Further, most clinicians received no training related to evaluation of DIMDs via telehealth or engaging caregivers as health care partners. Conclusion: Our study highlights specific limitations and challenges and provides considerations to help clinicians better manage DIMDs in the context of telehealth services.