Overexpression of the embryonic-lethal abnormal vision-like protein HuR in ovarian carcinoma is a prognostic factor and is associated with increased cyclooxygenase 2 expression

The human embryonic-lethal abnormal vision-like protein HuR is involved in the regulation of mRNA turnover and serves as a shuttling protein between the nucleus and the cytoplasm that stabilizes mRNAs containing adenine- and uridine-rich elements in their 3' untranslated region. We have shown recently that expression of cyclooxygenase (COX)-2 is related to poor prognosis in ovarian carcinoma. Other studies have shown that the COX-2 mRNA contains an adenine- and uridine-rich element and is stabilized by HuR. In this study, we investigated the expression and cellular distribution of HuR in 83 primary ovarian carcinomas, 16 borderline tumors of the ovary, 3 normal ovaries, and 9 ovarian carcinoma cell lines. Expression of HuR was detected in all cell lines on the mRNA and protein level and showed a predominantly nuclear staining in OVCAR-3 cells by confocal microscopy. In an immunohistochemical evaluation of human ovarian carcinomas, HuR showed a nuclear expression in 81% of tumors. In addition, a cytoplasmic expression of HuR was observed in a subgroup of 45% of ovarian carcinomas. Nuclear as well as cytoplasmic expression of HuR was significantly increased in ovarian carcinomas compared with borderline tumors or normal ovaries. In univariate analysis, a significant association between cytoplasmic HuR expression and increased COX-2 expression (P = 0.025) as well as between histological grade (P = 0.008) and mitotic activity (P = 0.002) was observed, although nuclear expression of HuR was not correlated with COX-2 expression or other clinicopathological parameters. In Kaplan-Meier survival analysis, increased cytoplasmic expression of HuR was a significant prognostic indicator for progression-free survival (P = 0.03) as well as overall survival (P = 0.007). In multivariate analysis using the Cox regression model, cytoplasmic expression of HuR was an independent prognostic parameter for reduced overall survival with a relative risk of 2.62 (95% confidence interval, 1.32-5.19). Our results suggest that there is a dysregulation of cellular distribution of the mRNA stability factor HuR in a subset of invasive ovarian carcinomas. This dysregulation appears to result in an increased expression of COX-2, an increased proliferative rate, and may lead to a reduced survival time. Additional studies are required to analyze the downstream effects of increased cytoplasmic expression of HuR. In addition, it would be interesting to investigate the prognostic role of increased cytoplasmic expression of HuR in prospective studies.     

SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant

BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.     

Cycle dependency of intrauterine vascular endothelial growth factor levels is correlated with decidualization and corpus luteum function

OBJECTIVE: To determine intrauterine levels of vascular endothelial growth factor (VEGF)-A during the menstrual cycle in the human female and to investigate the impact of decidualization and corpus luteum function. DESIGN: Prospective clinical study. SETTING: Tertiary university center. PATIENT(S): Fifty-four women with infertility problems. INTERVENTION(S): Intrauterine concentrations of VEGF-A were determined at various time points during the secretory phase using a novel intrauterine microdialysis device. Concomitantly, intrauterine insulin-like growth factor binding protein (IGFBP)-1 levels served as a paracrine parameter for decidualization. Serum progesterone (P) and E(2) levels were determined as markers for corpus luteum function.Intrauterine VEGF levels. RESULT(S): The VEGF levels in utero were clearly cycle dependent with increasing levels during the late secretory and premenstrual phases. There was a significant correlation with the decidualization marker IGFBP-1. In contrast, intrauterine VEGF levels showed a significant negative correlation with serum E(2) and P. CONCLUSION(S): Intrauterine VEGF levels are regulated in a cycle-dependent way. Increasing levels in the late secretory phase are clearly correlated with decidualization. In contrast, decreasing serum levels of steroids produced by the regressing corpus luteum are less likely to be responsible for increasing VEGF levels in the premenstrual phase.     

Sensitization of B-cell chronic lymphocytic leukemia cells to recombinant immunotoxin by immunostimulatory phosphorothioate oligodeoxynucleotides

A recombinant anti-CD25 immunotoxin, LMB-2, has shown clinical efficacy in hairy cell leukemia and T-cell neoplasms. Its activity in B-cell chronic lymphocytic leukemia (B-CLL) is inferior but might be improved if B-CLL cells expressed higher numbers of CD25 binding sites. It was recently reported that DSP30, a phosphorothioate CpG-oligodeoxynucleotide (CpG-ODN) induces immunogenicity of B-CLL cells by up-regulation of CD25 and other antigens. The present study investigated the antitumor activity of LMB-2 in the presence of DSP30. To this end, B-CLL cells from peripheral blood of patients were isolated immunomagnetically to more than 98% purity. Incubation with DSP30 for 48 hours augmented CD25 expression in 14 of 15 B-CLL samples, as assessed by flow cytometry. DSP30 increased LMB-2 cytotoxicity dose dependently whereas a control ODN with no CpG motif did not. LMB-2 displayed no antitumor cell activity in the absence of CpG-ODN as determined colorimetrically with an (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. In contrast, B-CLL growth was inhibited in 12 of 13 samples with 50% inhibition concentrations (IC(50)) in the range of LMB-2 plasma levels achieved in clinical studies. Two samples were not evaluable because of spontaneous B-CLL cell death in the presence of DSP30. Control experiments with an immunotoxin that does not recognize hematopoietic cells, and an anti-CD22 immunotoxin, confirmed that sensitization to LMB-2 was specifically due to up-regulation of CD25. LMB-2 was much less toxic to normal B and T lymphocytes compared with B-CLL cells. In summary, immunostimulatory CpG-ODNs efficiently sensitize B-CLL cells to a recombinant immunotoxin by modulation of its target. This new treatment strategy deserves further attention.     

The amputee mobility predictor: an instrument to assess determinants of the lower-limb amputee's ability to ambulate

OBJECTIVES: To describe the development of the Amputee Mobility Predictor (AMP) instrument designed to measure ambulatory potential of lower-limb amputees with (AMPPRO) and without (AMPnoPRO) the use of a prosthesis, and to test its reliability and validity. DESIGN: Measurement study using known groups method and concurrence with existing measures. SETTING: Academic medical center. PARTICIPANTS: A convenience sample of 191 lower-limb amputee subjects who had completed prosthetic training, 24 in the reliability study (mean age +/- standard deviation, 68.3+/-17.9y, range, 28-99y) and 167 in the validity study (mean age, 54.8+/-18.6y; range, 18-100y). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Intra- and interrater reliability; construct validity by known groups method; concurrent validity by comparisons with 6-minute walk test, Comorbidity Index, age, and time since amputation; predictive validity by comparison with 6-minute walk test after controlling for other factors. RESULTS: Interrater reliability was.99 for subjects tested with and without their prosthesis; intrarater reliability was.96 and.97. Both the AMPnoPRO (P<.0001) and the AMPPRO scores (P<.0001) distinguished among the 4 Medicare functional classification levels. The AMP correlated strongly with 6-minute walk scores (AMPnoPRO r=.69, P<.0001; AMPPRO r=.82, P<.0001) and the amputee activity survey (AMPnoPRO r=.67, P<.0001; AMPPRO r=.77, P<.0001), and negatively correlated with age (AMPnoPRO r=-.69, P<.0001; AMPPRO r=.56, P<.0001) and comorbidity (AMPnoPRO r=-.43, P<.0001; AMPPRO r=.38, P<.0001). CONCLUSION: The AMP with and without a prosthesis are reliable and valid measures for the assessment of functional ambulation in lower-limb amputee subjects.     

Incidental intracranial hemorrhage after uncomplicated birth: MRI before and after neonatal heart surgery

We investigated the prevalence of intracranial hemorrhage (ICH) before and after neonatal heart surgery. We carried out pre- and postoperative MRI looking for brain lesions in 24 full-term newborns with known congenital heart disease. They underwent heart surgery with cardiopulmonary bypass (CPB), usually with deep hypothermic circulatory arrest (DHCA). The first MRI was 1-22 days after birth. There were 21 children born after uncomplicated vaginal delivery and three delivered by cesarean section (CS). ICH was seen in 13 (62%) of the vaginal delivery group but in none of the CS group. We saw subdural bleeding along the inferior surface of the tentorium in 11 (52%) and supratentorially in six (29%) of the 21 children with ICH. Small hemorrhages were present in the choroid plexus in seven (33%), in the parenchyma in one (5%) and in the occipital horn in one (5%). There were 26 foci of bleeding in these 21 patients (1.2 per patient). None was judged by formal neurologic examination to be symptomatic from the hemorrhage. Follow-up MRI after cardiac surgery was obtained in 23 children, showing 37 foci of ICH (1.6 per patient), but all appeared asymptomatic. Postoperatively, ICH had increased in 10 children (43%), was unchanged in seven (30%) and was less extensive in six (26%).     

Cytotoxic effect of conjugates of doxorubicin and human chorionic gonadotropin (hCG) in breast cancer cells

Cytotoxic activity of drug conjugates of human chorionic gonadotropin (hCG) and doxorubicin alone was investigated compared to doxorubicin in breast cancer cells with and without expression of hCG receptors. Expression of hCG receptor was determined in MCF-7 and MB231 breast cancer cell line using a multiplex nested rt-PCR approach. The entire sequence of mRNA encoding for hCG receptor was detected in MCF-7 but not in MB231 breast cancer cell line. Cytostatic effect of doxorubicin–hCG conjugates was investigated in these cell lines in comparison to unconjugated doxorubicin. The number of viable cells was determined after 24, 48, 72, 96, and 120 h. To exclude non-specific uptake of the carrier hCG from the culture media, a similar experiment was performed with albumin–doxorubicin conjugates. The number of viable cells decreased in a concentration depending manner after doxorubicin and hCG–doxorubicin conjugate treatment. However, the cytotoxic effect of hCG–doxorubicin conjugate was 10-fold increased compared to unconjugated doxorubin in hCG-receptor positive MCF-7 but not in hCG-receptor negative MB231 cells. Albumin–doxorubicin conjugates showed no increased toxicity compared to doxorubicin. We conclude that the cytotoxic effect of hCG–doxorubicin conjugates is mediated specifically via the hCG receptor. By using hCG conjugates, the development of more selective cytostatics can be achieved.     

Standardized extracts of flavonoids increase the viability of PC12 cells treated with hydrogen peroxide: effects on oxidative injury

Oxidative stress plays an important role in cell death associated with many diseases. In the present study, concentration-dependence of hydrogen peroxide on rat pheochromocytoma (PC12) cell viability was studied. Preventive effects of antioxidants on the viability of these cells treated with 2 mM hydrogen peroxide were compared. Trolox and Stobadine, as chain-breaking antioxidants were studied in comparison with standardized extracts of flavonoids of Ginkgo biloba and Pycnogenol, known as agents effective in several diseases. All antioxidants increased the viability of hydrogen peroxide-treated PC12 cells. Flavonoid extracts were more effective than Trolox and Stobadine. Antioxidants were most effective if present after the oxidative treatment. As expected, the preloading with antioxidants was without effect on cell viability. Correlations between viability increase induced by antioxidants, and content of oxidation products of proteins and lipids were studied at concentrations of antioxidants mostly effective in preventing cell death: Trolox (10 microM), Stobadine (30 microM), Ginkgo biloba (160 microg/ml), Pycnogenol (100 microg/ml). In these concentrations, antioxidants did not statistically significantly decrease the content of protein carbonyls, with exception of Stobadine, which had no effect. Ginkgo biloba, Trolox and Stobadine intensively decreased the content of malondialdehyde, a product of lipid peroxidation. Pycnogenol was without any preventive effect. Concentrations of antioxidants with a large effect on viability of PC12 cells were not effective in preventing oxygen radical-induced injury of proteins. Antioxidants prevented the oxidative injury of lipids more effectively than that of proteins.     

Breast and cervical cancer survival: making sense of ‘league tables’

During 1998, the Department of Health proposed to use survival rates of cervical and breast cancer in the 1989/90 incidence cohort as indicators of care. Valid interpretation was of concern within Trent and the Trent Cancer Registry responded by performing additional analyses. Trent Cancer Registry registrations for 1989/90 were re-analysed and the stability of districts' ranks for that cohort was investigated using random simulation techniques. Stability of ranks across more recent cohorts was investigated and attempts made to use all available information.The Department of Health's analyses were confirmed by our re-analysis of the 1989/90 cohort: Rotherham residents appeared to have the "worst" survival for cervical cancer, and Sheffield residents for breast cancer, although not statistically significantly so. Random simulations indicated that ranks based on a single cohort are not stable: for example Sheffield (ranked tenth for 1-y breast cancer survival) was ranked third or better in 6% of randomisations. Ranks were also unstable across cohorts: for example Rotherham 1-y cervical cancer survival was ranked tenth for 1989/90, fifth for 1991/92 and tenth for 1993/94. Analysis of 3-y running averages provided better information than the league table approach. Most districts improved over time, to different degrees, and similar sized gaps remained between the "best" and the "worst" districts. This analysis illustrates the need to be circumspect when interpreting "league tables" based on a single year or cohort analysis. League tables are based on ranks: clearly a large difference in rank may reflect only trivial (ie medically unimportant) differences in actual outcome. Lack of a statistically significant difference in survival between two districts does not mean their survival is equivalent. Even for a common cancer, like breast cancer, rankings were unstable from cohort to cohort. At the Registry we propose to perform these trend analyses routinely in future, adjusting, when possible, for the effects of deprivation and stage at diagnosis.