Abrogation of the Postmitotic Checkpoint Contributes to Polyploidization in Human Papillomavirus E7-Expressing Cells

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.An important hallmark of human cancers is aneuploidy, the state in which a cell has extra or missing chromosomes (12, 25). Polyploidy is the state in which cells have more than two equal sets of chromosomes and is thought to be an early event in multistep carcinogenesis that can lead to aneuploidy (1, 24), as exemplified in Barrett''s esophagus (11). Polyploidy has recently been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy (26). Other recent studies have shown that tetraploid but not diploid mouse or human cells induce tumor formation in mice (3, 9). These studies highlight the potential importance of polyploidy in carcinogenesis.The cellular mechanisms responsible for this polyploidy formation are as of yet undetermined, but several models have been proposed. First, abrogation of the spindle assembly checkpoint followed by cleavage failure may lead to polyploidy formation (36, 40). A second proposed model is rereplication, a process of multiple rounds of DNA replication without an intervening mitosis. Third, cells that adapt to the mitotic spindle checkpoint halt in a G₁-like state with 4C DNA content. Abrogation of this postmitotic checkpoint allows the cells to replicate their 4C DNA content, leading to polyploidy formation. This has been shown in cells that express the human papillomavirus type 16 (HPV-16) E6 oncogene that degrades p53 (21). Finally, cleavage failure, which yields binucleate cells with 4C DNA content, is also a potential mechanism for polyploidy formation (31).The postmitotic checkpoint becomes activated when cells with an intact spindle assembly checkpoint become arrested during mitosis for a prolonged period of time and eventually adapt to the checkpoint, exit mitosis without cleavage, and progress into a G₁-like state with 4C DNA content (19, 22). The cells are prevented from continuing through the cell cycle and replicating their DNA by a proposed p53- and pRb-dependent postmitotic checkpoint (18, 19).High-risk types of HPV (of which HPV-16 is the most prevalent) are commonly associated with lesions that can progress to cervical carcinoma, which is one of the leading causes of cancer death in women worldwide (42). The transforming properties of high-risk HPVs primarily reside in the E6 and E7 oncogenes (reviewed in reference 7). The ability of high-risk HPV E6 and E7 proteins to promote the degradation of p53 and pRb, respectively, has been suggested as a mechanism by which HPV induces cellular transformation (6, 30). Expression of the high-risk HPV E6 and E7 oncogenes in human keratinocytes leads to polyploidy, which is enhanced by DNA damage and by activation of the spindle checkpoint through microtubule disruption (15, 27, 37, 38).Previously, it was thought but not directly shown that high-risk E6 and E7 induce polyploidy in response to microtubule disruption by abrogating the spindle checkpoint and that degradation of the tumor suppressor p53 by E6 is the mechanism by which E6 accomplishes this polyploidy formation (27, 37, 38). Others have proposed that E7 may play a role in stimulating DNA rereplication that occurs prior to mitosis initiation and polyploidy formation (20). Our recent studies demonstrate that E6 does not affect the mitotic spindle checkpoint (21). Instead, E6 abrogates the postmitotic checkpoint to induce polyploidy after microtubule disruption. Interestingly, E6 mutant proteins defective in inducing p53 degradation also induce polyploidy (21). The mechanism by which HPV E7 induces polyploidy remains to be determined. In this study, we investigate these possible mechanisms through which HPV-16 E7 induces polyploidy formation.     

Isotopic evidence for the provenance and turnover of organic carbon by soil microorganisms in the Antarctic dry valleys

The extremely cold and arid Antarctic dry valleys are one of the most environmentally harsh terrestrial ecosystems supporting organisms in which the biogeochemical transformations of carbon are exclusively driven by microorganisms. The natural abundance of ¹³C and ¹⁵N in source organic materials and soils have been examined to obtain evidence for the provenance of the soil organic matter and the C loss as CO₂ during extended incubation (approximately 1200 days at 10°C under moist conditions) has been used to determine the potential decay of soil organic C. The organic matter in soils remote from sources of liquid water or where lacustrine productivity was low had isotope signatures characteristic of endolithic (lichen) sources, whereas at more sheltered and productive sites, the organic matter in the soils that was a mixture mainly lacustrine detritus and moss-derived organic matter. Soil organic C declined by up to 42% during extended incubation under laboratory conditions (equivalent to 50–73 years in the field on a thermal time basis), indicating relatively fast turnover, consistent with previous studies indicating mean residence times for soil organic C in dry valley soils in the range 52–123 years and also with recent inputs of relatively labile source materials.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

PROTEOLIPID PROTEIN: SYNTHESIS AND ASSEMBLY INTO QUAKING MOUSE MYELIN

The incorporation of radioactive glycine into the major myelin proteolipid protein isolated from whole brain and from purified myelin of Quaking mice and normal littermates was compared. In a typical experiment, four Quaking mice and four littermate controls were injected intracranially with 250 μCi [2-³H]glycine and 25 μCi [U-¹⁴C]glycine respectively. Three hours later, the eight mice were killed and their brains combined. Equivalent portions were taken for (1) chloroform-methanol (2:1) extraction followed by ether precipitation of proteolipid from the brain and (2) myelin preparation. The ^3H/¹⁴C ratios for the microsomes:, the major myelin proteolipid as well as the other non-myelin proteolipids extracted from whole brain was approx 3.0. while the ³H/¹⁴C ratio for proteolipid protein in myelin was near 0.4. These findings were consistent for ages studied between 18 and 90 days. The results indicate that the synthesis of the major myelin proteolipid protein in the whole brain of Quaking mouse, as seen previously in our studies on basic protein, proceeds at a normal rate relative to microsomes but its incorporation into myelin is depressed. A working hypothesis of myelin membrane assembly is presented to account for the defect in the incorporation of these proteins into Quaking myelin.     

Long-term platinum retention after treatment with cisplatin and oxaliplatin

Background  

The aim of this study was to evaluate long-term platinum retention in patients treated with cisplatin and oxaliplatin.     

LIMaS: the JAVA-based application and database for microarray experiment tracking

Microarrays allow monitoring of gene expression for tens of thousands of genes in parallel and are being used routinely to generate huge amounts of valuable data. Handling and analysis of such data are becoming major bottlenecks in the utilization of the technology. To enable the researcher to interpret the results postanalysis, we have developed a laboratory information management system for microarrays (LIMaS) with an n-tier Java front-end and relational database to record and manage large-scale expression data preanalysis. This system enables the laboratory to replace the paper trail with an efficient and fully customizable interface giving it the ability to adapt to any working practice, e.g., handling many resources used to form many products (chaining of resources). The ability to define sets of activities, resources, and workflows makes LIMaS MIAME-supportive.LIMaS is available for download at (http://www.mgu.har.mrc.ac.uk/microarray.)