Loss of Nek11 Prevents G2/M Arrest and Promotes Cell Death in HCT116 Colorectal Cancer Cells Exposed to Therapeutic DNA Damaging Agents

The Nek11 kinase is a potential mediator of the DNA damage response whose expression is upregulated in early stage colorectal cancers (CRCs). Here, using RNAi-mediated depletion, we examined the role of Nek11 in HCT116 WT and p53-null CRC cells exposed to ionizing radiation (IR) or the chemotherapeutic drug, irinotecan. We demonstrate that depletion of Nek11 prevents the G2/M arrest induced by these genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage. Interestingly, Nek11 depletion also led to long-term loss of cell viability that was independent of p53 and exacerbated following IR exposure. CRC cells express four splice variants of Nek11 (L/S/C/D). These are predominantly cytoplasmic, but undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain. In HCT116 cells, Nek11S in particular has an important role in the DNA damage response. These data provide strong evidence that Nek11 contributes to the response of CRC cells to genotoxic agents and is essential for survival either with or without exposure to DNA damage.     

Systematic evaluation of candidate blood markers for detecting ovarian cancer

Background

Epithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer.

Methods and Findings

We selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers-MUC16, WFDC2, MSLN and MMP7-warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer.

Conclusions

By systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.     

Crystal structure of a bacterial cocaine esterase.

Here we report the first structure of a cocaine-degrading enzyme. The bacterial esterase, cocE, hydrolyzes pharmacologically active (-)-cocaine to a non-psychoactive metabolite with a rate faster than any other reported cocaine esterase (kcat = 7.8 s-1 and KM = 640 nM). Because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose. The crystal structure of cocE, solved by multiple anomalous dispersion (MAD) methods, reveals that cocE is a serine esterase composed of three domains: (i) a canonical alpha/beta hydrolase fold (ii) an alpha-helical domain that caps the active site and (iii) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site was identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which were refined at 1.58 A and 1.63 A resolution, respectively. These structural studies suggest that substrate recognition arises partly from interactions between the benzoyl moiety of cocaine and a highly evolved specificity pocket.     

Identification of a single missense mutation in the adenine phosphoribosyltransferase (APRT) gene from five Icelandic patients and a British patient.

We have completely sequenced the adenine phosphoribosyltransferase (APRT) gene from each of six patients--five (I-V) from Iceland and one (VI) from Britain. Cases I and II shared a common ancestor six and seven generations ago, and cases I and V shared a common ancestor seven generations ago, but cases III and IV were unrelated to the above or to each other, over seven generations. Genomic DNA was amplified by PCR, subcloned into M13mp18, and sequenced. Genomic and PCR-amplified DNAs were also analyzed by restriction-enzyme digestion and Southern blotting. The same missense mutation was identified in all six patients. This mutation leads to the replacement of asp (GAC) by val (GTC), at amino acid position 65. The gene sequences from all patients were otherwise identical to our wild-type sequence. The homozygous nature of the mutation was confirmed by sequencing the PCR product directly. All six patients were homozygous for the 1.25-kb TaqI RFLP. The Icelandic patients were also homozygous for the 8-kb SphI RFLP, but the British patient was heterozygous at this site. These studies suggest that a founder effect is likely to be responsible for APRT deficiency in the Icelandic population. The finding of the same mutation in a patient from Britain suggests that this mutation may have originated in mainland Europe.     

Effect of juvenile hormone on acid phosphatases in the degenerating flight muscles of the Douglas-fir beetle, Dendroctonus pseudotsugae

Changes in acid phosphatase activity were used to estimate flight muscle degeneration in Dendroctonus pseudotsugae. Acid phosphatase positive sites increased numerically as well as in size in flight muscles of beetles attacking the host logs and those maintained on host bark chips. Topical treatment of beetles with a juvenile hormone analogue further increased the phosphatase activity. Juvenile hormone produced similar effects when applied to insects injected with eserine. An increase in the activity of acid phosphatases was also observed after incubation in culture medium with added juvenile hormone. The rôle of nerves and hormones in the degenerating muscles of D. pseudotsugae is compared with that in the permanently degenerating muscles during metamorphosis of some moths.     

Tumour infiltrating lymphocytes correlate with improved survival in patients with oesophageal adenocarcinoma

Background

Oesophageal adenocarcinoma (OAC) is increasingly common in the west, and survival remains poor at 10–15 % at 5 years. Immune responses are increasingly implicated as a determining factor of tumour progression. The ability of lymphocytes to recognise tumour antigens provides a mechanism for a host immune attack against cancer providing a potential treatment strategy.

Materials and Methods

Tumour infiltrating lymphocytes (TILs: CD3+, CD4+, CD8+ and FOXp3+) were assessed by immunohistochemistry using tissue microarrays in a contemporary and homogeneous cohort of OAC patients (n = 128) undergoing curative treatment.

Results

Multivariate analysis identified three independent prognostic factors for improved cancer-specific survival (CSS): increased CD8+ TILs (p = 0.003), completeness of resection (p < 0.0001) and lower pathological N stage (p < 0.0001). Independent prognostic factors for favourable disease-free survival included surgery-only treatment (p = 0.015), completeness of resection (p = 0.001), increased CD8+ TILs (p < 0.0001) and reduced pathological N stage (p < 0.0001). Higher levels of TILs in the pathological specimen were associated with significant pathological response to neoadjuvant chemotherapy (NAC). On multivariate analysis increased levels of CD4+ (p = 0.017) and CD8+ TILs (p = 0.005) were associated with significant local tumour regression and lymph node downstaging, respectively.

Discussion

Our results establish an association of TILs and survival in OAC, as seen in other solid tumours, and identify particular TIL subsets that are present at higher levels in patients who responded to NAC compared to non-responders. These findings highlight potential therapeutic strategies in EAC based on utilising the host immunological response and highlight the immune responses biomarker potential.

     

Relative efficiency of N fertilizers as influenced by N-serve in the potato crop

Summary Field experiments, on potato, were conducted in the alluvial soils of Ludhiana, in 1976–77 and 1980–81, to investigate the relative efficiency of N fertilizers as influenced by a nitrification inhibitor (N-serve). In the absence of N-serve, sulphate of ammonia (S/A) and calcium ammonium nitrate (CAN) proved superior to urea, on an equal N basis, in almost all the yield characters and yield, but urea treated with N-serve tended to equal S/A and proved better than CAN. The inhibitor increased the N-uptake and N-recovery from urea and decreased the optimum basal dose of the fertilizer, by 10.8kg N/ha in 1976–77 and by 40.5 kg N/ha in 1980–81, without reducing the tuber yield in any of the two years. S/A and CAN did not profit from the treatment with N-serve.