Clathrin Is Spindle-Associated but Not Essential for Mitosis

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells

Measurement of serum total and free prostate-specific antigen in women with colorectal carcinoma

We investigated the diagnostic value and the relationship with clinicopathological features of total and free prostate-specific antigen by measuring the concentrations of these markers in the sera of 75 women with colorectal carcinoma and in 30 healthy women. Measurements were performed by immunoradiometric assay which utilizes monoclonal and polyclonal anti-prostate-specific antigen antibodies; the lowest detection level for both markers was 0.01 ng ml−1. Free prostate-specific antigen levels were significantly higher in women with colorectal carcinoma than healthy women (P=0.006). The percentage of free prostate-specific antigen predominant (free prostate-specific antigen/total prostate-specific antigen >50%) subjects was 20% in colorectal carcinoma patients and 3.3% in healthy women (P=0.035). Cut-off values were 0.34 ng ml−1 for total prostate-specific antigen and 0.01 ng ml−1 for free prostate-specific antigen. In women with colorectal carcinoma, total prostate-specific antigen positivity was 20% and free prostate-specific antigen positivity was 34.6%. When compared to negatives, total prostate-specific antigen positive patients had a lower percentage of well-differentiated (P=0.056) and early stage (stages I and II) tumours (P=0.070). However, patients with predominant free prostate-specific antigen, had a higher percentage of well-differentiated (P=0.014) and early stage tumours (P=0.090) than patients with predominant bound prostate-specific antigen. In conclusion, although the sensitivity of free prostate-specific antigen predominancy is low (20%), in distinguishing women with colorectal carcinoma than healthy women, its specificity is high (96.7%). Free prostate-specific antigen predominancy tends to be present in less aggressive tumours. These findings may indicate clinical significance of preoperative measurement of serum total and free prostate-specific antigen in women with colorectal carcinoma

Observation of an Excited Bc+ State

Using pp collision data corresponding to an integrated luminosity of 8.5 fb-1 recorded by the LHCb experiment at center-of-mass energies of s=7, 8, and 13 TeV, the observation of an excited Bc+ state in the Bc+π+π- invariant-mass spectrum is reported. The observed peak has a mass of 6841.2±0.6(stat)±0.1(syst)±0.8(Bc+) MeV/c2, where the last uncertainty is due to the limited knowledge of the Bc+ mass. It is consistent with expectations of the Bc∗(2S31)+ state reconstructed without the low-energy photon from the Bc∗(1S31)+→Bc+γ decay following Bc∗(2S31)+→Bc∗(1S31)+π+π-. A second state is seen with a global (local) statistical significance of 2.2σ (3.2σ) and a mass of 6872.1±1.3(stat)±0.1(syst)±0.8(Bc+) MeV/c2, and is consistent with the Bc(2S10)+ state. These mass measurements are the most precise to date

Multilineage hematopoietic recovery with concomitant antitumor effects using low dose Interleukin-12 in myelosuppressed tumor-bearing mice

<p>Abstract</p> <p>Background</p> <p>Interleukin-12 (IL-12) is a cytokine well known for its role in immunity. A lesser known function of IL-12 is its role in hematopoiesis. The promising data obtained in the preclinical models of antitumor immunotherapy raised hope that IL-12 could be a powerful therapeutic agent against cancer. However, excessive clinical toxicity, largely due to repeat dose regimens, and modest clinical response observed in the clinical trials have pointed to the necessity to design protocols that minimize toxicity without affecting the anti-tumor effect of IL-12. We have focused on the lesser known role of IL-12 in hematopoiesis and hypothesized that an important clinical role for IL-12 in cancer may be as an adjuvant hematological cancer therapy. In this putative clinical function, IL-12 is utilized for the prevention of cancer therapy-related cytopenias, while providing concomitant anti-tumor responses over and above responses observed with the primary therapy alone. This putative clinical function of IL-12 focuses on the dual role of IL-12 in hematopoiesis and immunity.</p> <p>Methods</p> <p>We assessed the ability of IL-12 to facilitate hematopoietic recovery from radiation (625 rad) and chemotherapy (cyclophosphamide) in two tumor-bearing murine models, namely the EL4 lymphoma and the Lewis lung cancer models. Antitumor effects and changes in bone marrow cellularity were also assessed.</p> <p>Results</p> <p>We show herein that carefully designed protocols, in mice, utilizing IL-12 as an adjuvant to radiation or chemotherapy yield facile and consistent, multilineage hematopoietic recovery from cancer therapy-induced cytopenias, as compared to vehicle and the clinically-utilized cytokine granulocyte colony-stimulating factor (G-CSF) (positive control), while still providing concomitant antitumor responses over and above the effects of the primary therapy alone. Moreover, our protocol design utilizes single, low doses of IL-12 that did not yield any apparent toxicity.</p> <p>Conclusion</p> <p>Our results portend that despite its past failure, IL-12 appears to have significant clinical potential as a hematological adjuvant cancer therapy.</p

Isotropic 3D Nuclear Morphometry of Normal, Fibrocystic and Malignant Breast Epithelial Cells Reveals New Structural Alterations

Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis

A randomized two arm phase III study in patients post radical resection of liver metastases of colorectal cancer to investigate bevacizumab in combination with capecitabine plus oxaliplatin (CAPOX) vs CAPOX alone as adjuvant treatment

<p>Abstract</p> <p>Background</p> <p>About 50% of patients with colorectal cancer are destined to develop hepatic metastases. Radical resection is the most effective treatment for patients with colorectal liver metastases offering five year survival rates between 36-60%. Unfortunately only 20% of patients are resectable at time of presentation. Radiofrequency ablation is an alternative treatment option for irresectable colorectal liver metastases with reported 5 year survival rates of 18-30%. Most patients will develop local or distant recurrences after surgery, possibly due to the outgrowth of micrometastases present at the time of liver surgery. This study aims to achieve an improved disease free survival for patients after resection or resection combined with RFA of colorectal liver metastases by adding the angiogenesis inhibitor bevacizumab to an adjuvant regimen of CAPOX.</p> <p>Methods/design</p> <p>The Hepatica study is a two-arm, multicenter, randomized, comparative efficacy and safety study. Patients are assessed no more than 8 weeks before surgery with CEA measurement and CT scanning of the chest and abdomen. Patients will be randomized after resection or resection combined with RFA to receive CAPOX and Bevacizumab or CAPOX alone. Adjuvant treatment will be initiated between 4 and 8 weeks after metastasectomy or resection in combination with RFA. In both arms patients will be assessed for recurrence/new occurrence of colorectal cancer by chest CT, abdominal CT and CEA measurement. Patients will be assessed after surgery but before randomization, thereafter every three months after surgery in the first two years and every 6 months until 5 years after surgery. In case of a confirmed recurrence/appearance of new colorectal cancer, patients can be treated with surgery or any subsequent line of chemotherapy and will be followed for survival until the end of study follow up period as well. The primary endpoint is disease free survival. Secondary endpoints are overall survival, safety and quality of life.</p> <p>Conclusion</p> <p>The HEPATICA study is designed to demonstrate a disease free survival benefit by adding bevacizumab to an adjuvant regime of CAPOX in patients with colorectal liver metastases undergoing a radical resection or resection in combination with RFA.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov Identifier NCT00394992</p

Distinct HR expression patterns significantly affect the clinical behavior of metastatic HER2+ breast cancer and degree of benefit from novel anti-HER2 agents in the real world setting

We analyzed data from 738 HER2\u2010positive metastatic breast cancer (mbc) patients treated with pertuzumab\u2010based regimens and/or T\u2010DM1 at 45 Italian centers. Outcomes were explored in relation to tumor subtype assessed by immunohistochemistry (IHC). The median progression free survival at first\u2010line (mPFS1) was 12 months. Pertuzumab as first\u2010line conferred longer mPFS1 compared to other first\u2010line treatments (16 vs 9 months, p=0.0001), regardless of IHC subtype. Median PFS in second\u2010line (mPFS2) was 7 months, with no difference by IHC subtype, but it was more favorable with T\u2010DM1 compared to other agents (7 vs 6 months, p=0.03). There was no PFS2 gain in patients with tumors expressing both hormonal receptors (HRs) (p=0.17), while a trend emerged for tumors with one HR (p=0.05). Conversely, PFS2 gain was significant in HRs\u2010negative tumors (p=0.04). Median overall survival (mOS) was 74 months, with no significant differences by IHC subtypes. Survival rates at 2 and 3 years in patients treated with T\u2010DM1 in second\u2010line following pertuzumab were significantly lower compared to pertuzumab\u2010na\uefve patients(p=0.01). When analyzed by IHC subtype, the outcome was confirmed if both HRs or no HRs were expressed (p=0.02 and p=0.006, respectively). Our results confirm that HRs expression impacts the clinical behavior and novel treatment\u2010related outcomes of HER2\u2010positive tumors when treatment sequences are considered. Moreover, multivariate analysis showed that HRs expression had no effect on PFS and OS. Further studies are warranted to confirm our findings and clarify the interplay between HER2 and estrogen receptor (ER) pathways in HER2\u2010positive (mbc) patients