Temporal integration characteristics of the axial and choroidal responses to myopic defocus induced by prior form deprivation versus positive spectacle lens wear in chickens.

PURPOSE: In chicks, the temporal response characteristics to form deprivation and to spectacle lens wear (myopic and hyperopic defocus) show essential differences, suggesting that the emmetropization system "weights" the visual signals differently. To further explore how the eye integrates opposing visual signals, we examined the responses to myopic defocus induced by prior form deprivation vs. that induced by positive spectacle lenses, in both cases alternating with form deprivation. METHODS: Three experimental paradigms were used: 1) Form deprivation was induced by monocular occluders for 7 days. Over the subsequent 7 days, the occluders were removed daily for 12 hours (n = 13), 4 hours (n = 7), 2 hours (n = 7), or 0 hours (n = 6). 2) Birds were form-deprived on day 12. Over the subsequent 7 days, occluders were replaced with a +10 D lens for 2 hours per day (n = 13). 3) Starting at day 11, a +10 D lens was placed over one eye for 2 hours (n = 13), 3 hours (n = 5), or 6 hours (n = 10) per day and were otherwise untreated. Ocular dimensions were measured with high-frequency A-scan ultrasonography; refractive errors were measured by streak retinoscopy at various intervals. RESULTS: In recovering eyes, 2 hours per day of myopic defocus was as effective as 12 hours at inducing refractive and axial recovery (change in refractive error: +10 D vs. +13 D, respectively). By contrast, 2 hours of lens-induced defocus (alternating with form deprivation) was not sufficient to induce refractive or axial compensation (change in refractive error: -1.7 D). When myopic defocus alternated with unrestricted vision, 6 hours per day were sufficient to induce nearly full compensation (2 hours vs. 6 hours: 4.4 D vs. 8.2 D; p < 0.0005). Choroids showed rapid increases in thickness to the daily episodes of myopic defocus; these resulted in "long-term" thickness changes in recovering eyes and eyes wearing lenses for 3 or 6 hours per day. CONCLUSIONS: The response to myopic defocus induced by prior form deprivation is more robust than the response induced by positive lenses, suggesting that the underlying mechanisms differ. Presumably, this difference is related to the size of the eye at the onset. Compensatory decreases in growth rate occur without full compensatory choroidal thickening.     

CXCL13 is highly produced by Sézary cells and enhances their migratory ability via a synergistic mechanism involving CCL19 and CCL21 chemokines

Chemokine and chemokine receptors expressed by normal and neoplastic lymphocytes play a key role in cell recruitment into skin and lymph nodes. The aim of this study was to get further insights into the role of chemokines in pathogenesis and progression of cutaneous T-cell lymphoma (CTCL) with particular regard to Sézary Syndrome (SS), a CTCL variant with blood involvement. Here, we show that functional CXCL13 homeostatic chemokine is strongly up-regulated in SS cells, well-detectable in skin lesions and lymph nodes, and measurable at high concentration in plasma of SS patients, at different levels during disease progression. Furthermore, we show that the addition of CXCL13 to CCL19 or to CCL21, the selective CCR7 agonists responsible for lymph node homing, strongly enhances the migration of CCR7+ SS cells. We also show that neutralization of the CCR7 receptor strongly impairs CCL19/21-induced chemotaxis of SS cells both in the absence or presence of CXCL13. Additional experiments performed to investigate the survival, adhesion, and metalloproteases secretion indicate that CXCL13 combined with CCL19 and CCL21 mainly affects the chemotaxis of SS cells. Our findings suggest that this newly described CXCL13 expression in SS represents a new pathogenetic mechanism of diagnostic significance.     

Spontaneous T cell responses to Epstein-Barr virus-encoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy

Immunotherapy approaches targeting Epstein-Barr virus (EBV)-encoded antigens induce objective clinical responses only in a fraction of patients with undifferentiated nasopharyngeal carcinoma (UNPC). In the present study, we have characterized the immunogenicity of the EBV-encoded BARF1 oncogene with the aim to assess whether this protein could constitute a new target antigen for immunotherapy in this setting. Spontaneous CD4+ and CD8+ T cell responses specific for the recombinant p29 BARF1 protein were detected by IFNgamma-ELISPOT in both EBV-seropositive donors and UNPC patients, but not in EBV-seronegative individuals. Using immunoinformatic prediction tools, we have selected 5 different candidate BARF1 T cell epitopes presented by HLA-A*0201. Although only one of these peptides was able to bind HLA-A2 with low affinity in the T2 stabilization assay, all 5 BARF1 nonamers readily elicited specific CD8+ T cell responses in EBV-seropositive HLA-A*0201+ donors and UNPC patients. Notably, the magnitude of CD8+ T cell responses to the whole BARF1 protein and derived A*0201 peptides was significantly higher in UNPC patients than in healthy donors. Moreover, cytotoxic T lymphocytes specific for the p2-10, p23-31, or p49-57 BARF1 peptides were easily obtained from HLA-A*0201+ donors. These cultures were not only able to lyse autologous targets loaded with the antigenic peptide, but also recognized tumor cells endogenously expressing BARF1 in an antigen-specific and HLA-A2-restricted manner. These findings, indicate that BARF1 is a particularly attractive antigen with immunogenic properties in most UNPC patients and provide valuable information to develop new strategies to improve the efficacy of EBV-targeting immunotherapy of UNPC patients.     

Gene profiling assay and application: the predictive role in primary therapy

Several treatment options, including endocrine therapy, chemotherapy, and targeted therapy, have been shown to improve survival of breast cancer patients. Currently, clinical tests for predicting cancer response are not available, and individual markers have shown little predictive value. Several gene expression profiling studies have been carried out in the attempt to identify predictive signatures. The neoadjuvant setting revealed to be ideal for this purpose because it allows the direct assessment of response to treatment, and tumor is readily available for multiple time point biopsies. Although the results are promising, at the moment, none of these signatures has been proven to be of sufficient discriminatory power to be used in clinical setting. More effective therapies targeted to specific subsets of patients, accurate and standardized definition of therapeutic response, and properly designed clinical trials are required before microarrays can reliably be used as tools for clinical decision making.     

A cancer‐associated CDKN1B mutation induces p27 phosphorylation on a novel residue: a new mechanism for tumor suppressor loss‐of‐function

CDKN1B haploinsufficiency promotes the development of several human cancers. The gene encodes p27^Kip1, a protein playing pivotal roles in the control of growth, differentiation, cytoskeleton dynamics, and cytokinesis. CDKN1B haploinsufficiency has been associated with chromosomal or gene aberrations. However, very few data exist on the mechanisms by which CDKN1B missense mutations facilitate carcinogenesis. Here, we report a functional study on a cancer‐associated germinal p27^Kip1 variant, namely glycine9‐>arginine‐p27^Kip1 (G9R‐p27^Kip1) identified in a parathyroid adenoma. We unexpectedly found that G9R‐p27^Kip1 lacks the major tumor suppressor activities of p27^Kip1 including its antiproliferative and pro‐apoptotic functions. In addition, G9R‐p27^Kip1 transfection in cell lines induces the formation of more numerous and larger spheres when compared to wild‐type p27^Kip1‐transfected cells. We demonstrated that the mutation creates a consensus sequence for basophilic kinases causing a massive phosphorylation of G9R‐p27^Kip1 on S12, a residue normally never found modified in p27^Kip1. The novel S12 phosphorylation appears responsible for the loss of function of G9R‐p27^Kip1 since S12AG9R‐p27^Kip1 recovers most of the p27^Kip1 tumor suppressor activities. In addition, the expression of the phosphomimetic S12D‐p27^Kip1 recapitulates G9R‐p27^Kip1 properties. Mechanistically, S12 phosphorylation enhances the nuclear localization of the mutant protein and also reduces its cyclin‐dependent kinase (CDK)2/CDK1 inhibition activity. To our knowledge, this is the first reported case of quantitative phosphorylation of a p27^Kip1 variant on a physiologically unmodified residue associated with the loss of several tumor suppressor activities. In addition, our findings demonstrate that haploinsufficiency might be due to unpredictable post‐translational modifications due to generation of novel consensus sequences by cancer‐associated missense mutations.

Abbreviations

1D/WB

monodimensional western blotting

2D/WB

two‐dimensional western blotting

CDK

cyclin‐dependent kinase

CHX

cycloheximide

G9R‐p27

glycine9‐>arginine‐p27

IUPs

intrinsically unstructured proteins

mAbs

monoclonal antibodies

MEN

multiple endocrine neoplasia

MENX

multiple endocrine neoplasia X

PTMs

post‐translational modifications

rAbs

rabbit antibodies

TSG

tumor suppressor gene

wt‐p27

wild‐type p27

     

Combination effects of platinum drugs and N 1, N 11 diethylnorspermine on spermidine/spermine N 1-acetyltransferase, polyamines and growth inhibition in A2780 human ovarian carcinoma cells and their oxaliplatin and cisplatin-resistant variants

Purpose

To understand the mechanisms behind platinum drug/DENSPM-induced inhibition of cancer cell growth, we compared the effects of oxaliplatin and cisplatin when combined with DENSPM on the induction of SSAT mRNA, activity, polyamines and cell growth in A2780 human ovarian carcinoma cells and their oxaliplatin- and cisplatin-resistant variants A2780/C10B and A2780/CP, respectively.

Methods

Parental and Pt-resistant cells were treated with platinum agent alone, DENSPM alone or combination (10???M each, 20?h). QRT?CPCR, radioactive product measurement and HPLC were used for mRNA, activity and polyamine pools, respectively; drug interaction on cell growth was by SRB and isobologram analysis.

Results

Both platinum agents induced SSAT mRNA in parental A2780 cells, but not in resistant cells. Platinum drug/DENSPM combinations produced high levels of SSAT activity in parental cells with significant depletion of spermine and spermidine, but not in resistant cells. Co-treatment with platinum agents increased the levels of DENSPM in all cell lines. Oxaliplatin/DENSPM combination was superior to cisplatin/DENSPM in the inhibition of cell growth in parental cells. No synergy was observed in the resistant cells.

Conclusions

Increased DENSPM levels following co-treatment with Pt agents enhances the translation and stability of SSAT protein leading to polyamine pool depletion, facilitating more Pt?CDNA adduct formation in parental cells. Oxaliplatin/DENSPM combination is superior to cisplatin/DENSPM in cell growth inhibition as DACH-Pt DNA adducts are cytotoxic even at relatively fewer numbers. Reduced platinum uptake in Pt-resistant cells contributes to reduced SSAT mRNA induction and absence of synergy when combined with DENSPM.     

Identification of candidate genes carrying polymorphisms associated with the risk of colorectal cancer by analyzing the colorectal mutome and microRNAome

BACKGROUND:

The presence of single‐nucleotide polymorphisms (SNPs) within the 3′‐untranslated regions of genes could affect the binding between a microRNA (miRNA) and its target, with consequences on gene expression regulation. Considering the important role of miRNAs in carcinogenesis, it is hypothesized here that these SNPs could also affect the individual risk of colorectal cancer (CRC).

METHODS:

To test this hypothesis, a list was developed of 140 somatically mutated genes deduced from previous works on the mutome of the CRC. A further selection was conducted of SNPs within target sites for miRNAs that are expressed only in the colorectum (the colorectal microRNAome) and having adequate population frequencies. This yielded 12 SNPs that were genotyped in a case‐control association study on 717 colorectal cases and 1171 controls from the Czech Republic.

RESULTS:

Statistically significant associations were found between the risk of CRC and the variant alleles of KIAA0182 (rs709805) (odds ratio = 1.57; 95% confidence interval = 1.06‐2.78, for the variant homozygotes) and NUP210 genes (rs354476) (odds ratio = 1.36; 95% confidence interval = 1.02‐1.82, for the variant homozygotes).

CONCLUSIONS:

The results support the study hypothesis and highlight the importance of SNPs within miRNA‐dependent regulatory regions. Further studies on the role exerted by NUP210 and KIAA0182 in colorectal carcinogenesis are warranted. Cancer 2012. © 2012 American Cancer Society.     

Role of radiotherapy in the treatment of cervical lymph node metastases from an unknown primary site: retrospective analysis of 113 patients

PURPOSE: The management of patients with cervical lymph-node metastases from unknown primary site (UPS) remains a matter of discussion. This study aimed to analyze the results and prognostic factors in a series of patients treated with radiotherapy. METHODS AND MATERIALS: Data from 113 patients who presented with cervical lymph nodes metastases from UPS treated from 1980 to 2004 were reviewed. Eighty-seven patients (77.0%) were squamous cell carcinoma (SCC). Ninety-one patients were treated with curative and 22 with palliative intent. Fifty-nine of 113 patients (52.2%) received surgery followed by radiotherapy and 54 of 113 (47.8%) received radiotherapy alone. Radiotherapy was delivered to the neck and pharyngeal mucosa in 67 patients and to the ipsilateral or bilateral neck in 45 patients. Twenty-one patients (18.5%) also received chemotherapy. RESULTS: The 5-year overall survival rates were 40.7% for the entire group and 46.6% for the SCC subgroup. The occurrence of the occult primary was observed in 23 of 113 patients (20.3%), 19 (82.6%) within the head and neck region. At multivariate analysis, treatment with curative intent and extensive irradiation of bilateral neck and pharyngeal mucosa were favorable prognostic factors for the whole series, and treatment with curative intent, extensive irradiation of bilateral neck and pharyngeal mucosa, and absence of extracapsular spread were favorable prognostic factors for the SCC subgroup. CONCLUSIONS: Patients with cervical lymph node metastases from UPS have a similar prognosis to those affected by other head and neck malignancies. Curative treatment strategies including neck dissection and extensive irradiation by three-dimensional conformal radiation therapy resulted in significantly better outcomes.