Disturbances In Cell Cycle Regulation Research Articles

Heterogeneities in Cell Cycle Checkpoint Activation Following Doxorubicin Treatment Reveal Targetable Vulnerabilities in TP53 Mutated Ultra High-Risk Neuroblastoma Cell Lines.

Most chemotherapeutics target DNA integrity and thereby trigger tumour cell death through activation of DNA damage responses that are tightly coupled to the cell cycle. Disturbances in cell cycle regulation can therefore lead to treatment resistance. Here, a comprehensive analysis of cell cycle checkpoint activation following doxorubicin (doxo) treatment was performed using flow cytometry, immunofluorescence and live-cell imaging in a panel of TP53 mutated ultra high-risk neuroblastoma (NB) cell lines, SK-N-DZ, Kelly, SK-N-AS, SK-N-FI, and BE(2)-C. Following treatment, a dose-dependent accumulation in either S- and/or G2/M-phase was observed. This coincided with a heterogeneous increase of cell cycle checkpoint proteins, i.e., phos-ATM, phos-CHK1, phos-CHK2, Wee1, p21Cip1/Waf1, and p27Kip among the cell lines. Combination treatment with doxo and a small-molecule inhibitor of ATM showed a delay in regrowth in SK-N-DZ, of CHK1 in BE(2)-C, of Wee1 in SK-N-FI and BE(2)-C, and of p21 in Kelly and BE(2)-C. Further investigation revealed, in all tested cell lines, a subset of cells arrested in mitosis, indicating independence on the intra-S- and/or G2/M-checkpoints. Taken together, we mapped distinct cell cycle checkpoints in ultra high-risk NB cell lines and identified checkpoint dependent and independent druggable targets.

Radiation resistance: Cancer stem cells (CSCs) and their enigmatic pro-survival signaling.

Despite the fact that radiation therapy is a highly effective therapeutic approach, a small intratumoral cell subpopulation known as "cancer stem cells" (CSCs) is radiation-resistant and possesses specific molecular properties protecting it against radiation-induced damage. The exact mechanisms of this radioresistance are still not fully elucidated, but they relate to these cells' enhanced DNA repair capacities and their low intracellular ROS concentrations, resulting from their up-regulation of ROS scavengers. The low ROS content is accompanied by disturbances in cell cycle regulation, so it can be assumed that either CSCs are quiescent or dormant themselves, or that this cell population consists of at least two cell subpopulations: the normally and the slowly proliferating cells (quiescent or dormant cells). Slowly dividing CSCs show concomitant dysregulation of the signaling molecules mediating both cell cycle progression and maintenance of cell stemness. Despite a massive accumulation of data concerning the mechanisms underlying DNA damage response in CSCs, it represents a challenge to researchers in the era of personalized medicine to elucidate the role of intracellular ROS and of signaling pathways associated with the radiation resistance of these cells; there is a clear need to understand the molecular mechanisms helping CSCs to survive radiation exposure.

Swedish amyloid precursor protein mutation increases cell cycle‐related proteins in vitro and in vivo

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease. In this study, we report that the expressions of Swedish double mutation of amyloid precursor protein (Swe-APP) or of the APP intracellular domain (AICD) into nerve growth factor (NGF)-differentiated PC12 cells or rat primary cortical neurons increased mRNA and protein levels of cyclin D1 and cyclin B1. Treatment with lithium chloride (a glycogen synthase kinase-3beta inhibitor) down-regulated cyclin B1 induced by Swe-APP expression but up-regulated cyclin D1 expression induced by Swe-APP, suggesting that glycogen synthase kinase-3beta activity is involved in these expression changes of cyclins D1 and B1. Swe-APP, which is a prevailing cause of familial Alzheimer's disease, is well known to increase amyloid beta peptide production both in vitro and in vivo, but the underlying molecular means whereby it leads to the pathogenesis of AD remains unknown. The finding that cyclin D1 and B1 expressions were up-regulated by Swe-APP in in vitro cultured cells was substantiated in the brain tissues of Tg2576 mice, which harbor the Swe-APP mutation. These results suggest that some disturbances in cell cycle regulation may be involved in Swe-APP or AICD-induced neurodegeneration and that these contribute to the pathogenesis of AD.

Morphologie des Chiasma opticum bei Albinismus

Albinism is associated with a misrouting of fibers at the optic chiasm where the majority of fibers cross to the contralateral side. The cause of this abnormal decussation pattern reflects a disturbance of cell cycle regulation in the development of the retina which is in part controlled by melanin. Growing axons from retinal ganglion cells therefore arrive later than usual at the optic chiasm and are misrouted contralaterally. This atypical decussation leads to morphological changes of the optic chiasm including a reduced chiasm width with larger angles between optic nerves and tracts which can be shown by magnetic resonance imaging.

X chromosomal and autosomal loss of heterozygosity and microsatellite instability in human cervical carcinoma

The study analyzes tumor material and normal tissue from 27 patients with pure squamous cell carcinoma of the uterine cervix for loss of heterozygosity (LOH) and microsatellite instability (MSI) on 14 autosomal and 11 X chromosomal loci. Overall, 4-40% of the informative cases showed LOH at autosomal regions with the highest frequency at 3p (21-40%) and a marked frequency at 2q35-q37.1 (12.5%) and 17p13.3 (10%), representing regions with putative tumor suppressor gene (TSG) function. The frequency of X chromosomal LOH ranged from 4% to 20%, with a maximum at Xq28 (20%) and Xq11.2-q12 (17%), again indicating alterations in TSG. A 12% LOH was seen at Xq21.33-q22.3, a region encoding a protein with a regulatory function in the cell cycle via cyclin-dependent kinases. MSI was detected in autosomal regions in up to 7% in regions linked to the X chromosome in up to 11%, probably indicating alterations of mismatch repair mechanisms. Our results and those obtained from the literature suggest that autosomal LOH and MSI in carcinomas of the cervix uteri are predominantly found at regions with putative TSG function. Beside TSG alterations, X chromosomal LOH is probably more strongly connected to disturbances in cell cycle regulation.

X chromosomal and autosomal loss of heterozygosity and microsatellite instability in human cervical carcinoma

The study analyzes tumor material and normal tissue from 27 patients with pure squamous cell carcinoma of the uterine cervix for loss of heterozygosity (LOH) and microsatellite instability (MSI) on 14 autosomal and 11 X chromosomal loci. Overall, 4–40% of the informative cases showed LOH at autosomal regions with the highest frequency at 3p (21–40%) and a marked frequency at 2q35-q37.1 (12.5%) and 17p13.3 (10%), representing regions with putative tumor suppressor gene (TSG) function. The frequency of X chromosomal LOH ranged from 4% to 20%, with a maximum at Xq28 (20%) and Xq11.2-q12 (17%), again indicating alterations in TSG. A 12% LOH was seen at Xq21.33-q22.3, a region encoding a protein with a regulatory function in the cell cycle via cyclin-dependent kinases. MSI was detected in autosomal regions in up to 7% in regions linked to the X chromosome in up to 11%, probably indicating alterations of mismatch repair mechanisms. Our results and those obtained from the literature suggest that autosomal LOH and MSI in carcinomas of the cervix uteri are predominantly found at regions with putative TSG function. Beside TSG alterations, X chromosomal LOH is probably more strongly connected to disturbances in cell cycle regulation.

Analysis of radiation-inducible hSNK gene in human thyroid cells

The late effect of radiation on thyroid tumorigenesis is closely associated with the disturbance of cell cycle regulation. The polo-like kinase (PLKs) family plays an important role in several stages of mitosis. Using the cDNA subtraction method, we recently identified in irradiated cultured human thyroid cells a gene belonging to the PLKs family, the human homologue of mouse serum-inducible kinase (hSNK). Study of the gene structure revealed the presence of 14 exons spanning over 6 kb of genomic DNA, that encoded a 2.9 kb mRNA product ubiquitously expressed to a various extent in different human tissues. The 5′-flanking region of hSNK was examined to study the basic transcriptional activity of the gene. The core promoter with activation elements including the TATA-like motif and GC box resided within the first 120 nucleotides, upstream of an analogous transcription start site. Functional mapping of the regulatory region demonstrated the presence of both enhancer and repressor fragments. Analysis of 0.7 kb of the 5′-upstream region of the gene revealed a number of motifs comprising of putative binding sites for several transcription factors and, notably, the p53 binding homology sequence. In conclusion, the hSNK may be a unique target molecule to influence cell fate following irradiation.

Disturbances in hepatic cell-cycle regulation in mice with assembly-deficient keratins 8/18

Simple epithelial tissues such as liver and pancreas express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins. K8 and K18 null mice and transgenic mice that express mutant K18 (K18C) manifest several hepatocyte abnormalities and demonstrate that K8/18 are important in maintaining liver tissue and cell integrity, although other potential functions remain uncharacterized. Here, we report an additional abnormal liver phenotype, which is similar in K8 null, K18 null, and K18C mouse models. Liver histologic examination showed large polynuclear areas that lacked cell membranes, desmosomal structures, and filamentous actin. Similar, but less prominent, areas were observed in the pancreas. The parenchyma outside the polynuclear areas displayed irregular sinusoidal structures and markedly enlarged nuclei. Most K8 null hepatocytes were positive for the proliferating cell nuclear antigen (PCNA) with a doubled DNA content in comparison with the predominantly PCNA-negative wild-type hepatocytes. The distribution of the 14-3-3[zeta ] protein was also altered in K8 null mice. Taken together, our results indicate that absence of keratin filaments causes disturbances in cell-cycle regulation, driving cells into the S-G2 phase and causing aberrant cytokinesis. These effects could stem from disturbed functions of K8/18-dependent cell-cycle regulators, such as the signaling integrator, 14-3-3. (HEPATOLOGY 2001;34:1174-1183.)