Specific inhibition of basal mitogen-activated protein kinases and phosphatidylinositol 3 kinase activities in leukemia cells: a possible therapeutic role for the kinase inhibitors.

OBJECTIVE: The roles of phosphatidylinositol 3 (PI3K) and mitogen-activated protein kinases (MAPK) have been widely studied in terms of the differentiation process induced by several drugs (phorbol ester, vitamin D-3, retinoic acid, etc.), but their exact functions in leukemic cells' phenotype and their potential therapeutic role remain incompletely clarified. MATERIALS AND METHODS: In order to investigate this query, leukemia cells were cultured in presence of kinase inhibitors (KIs). Proliferation, apoptosis, and differentiation were analyzed at the cellular and molecular levels, using flow cytometry and reverse transcriptase quantitative polymerase chain reaction. RESULTS: SB203580, a P38 MAPK inhibitor, had no effect on cell proliferation, whereas LY294002, a PI3K inhibitor, and PD098059, a selective inhibitor of mitogen-activated extracellular regulated kinase (MEK) phosphorylation, arrested cells in G(0)/G(1). However, LY294002 and PD098059 acted using different mechanisms: LY294002 decreased the expression of phosphorylated S6RP, whereas PD098059 increased P21/waf1 antigen expression. SP600125, an inhibitor of N-terminal c-jun kinases, arrested cells in G(2) and induced an endoreplicative process. SP600125 increased p21 at both the mRNA and protein levels. G(2) blockage is dependent on the PI3K pathway and the endoreplicative process is dependent on the PI3K and extracellular regulated kinase (ERK) pathways and mRNA synthesis. On the other hand, PD098059 potentiated the apoptotic process induced by either SP600125 or LY294002. Modulation of the expression of CD11, CD15, CD18, and CD54 was cell-dependent. CONCLUSION: Our results suggest that KIs modulate proliferation of leukemia cells and that the MEK/ERK inhibitor, PD098059, in combination with either SP600125 or LY294002, could have clinical value.

Resistance to phorbol ester-induced differentiation in human myeloid leukemia cells: a hypothetic role for the mRNA stabilization process.

UM384 cells, derived from the human myeloid leukemia U937 cell line, fail to differentiate in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Using cDNA microarray and real-time quantitative PCR (RT-QPCR) approaches, we observed a difference in the response to TPA treatment: all the genes from U937 cells were continuously modulated from 2 to 24h. In UM384 cells, 60% of the genes were transiently modulated at 2h, then returned to control levels at 24h. Moreover, HuR, an AU-rich element-binding protein (ARE-BP), was differentially located in the two cell lines. Therefore, a defect of mRNA stabilization could be responsible for the resistance of UM384 cells to TPA-induced differentiation, suggesting a possible role for the post-transcriptional regulation in the leukemogenesis.

Is interferon gamma one key of metastatic potential increase in human bladder carcinoma?

PURPOSE: IFN-gamma is detected in the urine of bladder cancer patients after intravesical bacillus Calmette Guerin instillation. Because it acts in the anticancer process, we studied its cellular and molecular mechanisms of action on human bladder cancer cell lines. RESULTS: IFN-gamma (>5 ng.ml(-1))(>400 IU.ml(-1)) inhibited the growth of bladder cancer cell lines and modified the expression of the tumor-associated markers tissue-type plasminogen activators, Plasminogen activator inhibitor-2, urokinase plasminogen activator receptor, colony-stimulating factor 1, intercellular adhesion molecule 1, and class II MHC. Interestingly, IFN-gamma-induced apoptosis of the low-grade bladder cancer cell lines (RT4/G1 and RT112/G2) related to a cleavage of caspases 1, 8, and 9. This process was inhibited by the phosphatidylinositol 3'-kinase inhibitor (LY294002) and the protein synthesis inhibitor (cycloheximide). Moreover, low doses of IFN-gamma (<5 ng.ml(-1))(<400 IU.ml(-1)) increased the resistance to the cytotoxic effect of tumor necrosis factor alpha in the RT112 cells but not in the RT4 cells. This acquired resistance was associated with morphological changes and with an increase of the cell migration and scattering. CONCLUSIONS: We demonstrated that in the low-grade bladder cancer cell lines, the effect of IFN-gamma was dose dependent: high doses (>5 ng.ml(-1)) induced apoptosis of RT4 and RT112 cells, whereas low doses (<5 ng.ml(-1)) induced a resistance to the cytotoxic effect of tumor necrosis factor alpha and increase the metastatic potential of the RT112 cells. Therefore, we propose that a similar phenomenon could participate to the immunotherapy failure observed during tumor progression of bladder cancer.

Intra-serous haematopoiesis.

The samples from pleural and pericardial effusion, in which immature haematopoietic cells had been identified cytologically, were re-examined. The results were then analysed along with the clinico-biological context and compared to published data. The aim was to determine the frequency, the type and the context of haematopoietic cell identification in pleural and pericardial fluid effusion. In 10 years, 28 cases were studied. Four sub-groups were described: 1) patients with a severe sepsis, 2) patients with an acute local or regional infection, 3) persistent or recurrent effusion without specific context, 4) patients who underwent a transplantation treated with cyclosporin A. Even when the clinico-pathological context did not suggest a classical extra-medullary haematopoiesis, it was not exceptional to identify immature haematopoietic cells. This hypothesis is supported by published data, which suggests that a local inflammatory state could help mesothelial cells to constitute a favourable environment for division and maturation of circulating haematopoietic progenitor cells.

Fibrinogen mediates bladder cancer cell migration in an ICAM-1-dependent pathway.

Fibrinogen (fg), present in tumor matrices, has been demonstrated to be determinant in metastatic potential. We have recently shown that fg/ICAM-1 interactions are involved in leukocyte migration across endothelial cell monolayers. Using bladder transitional cell carcinoma as a model, we will show in this study that bladder high grade tumor cell lines express ICAM-1, and that this expression induces an fg-mediated migration. This phenomenon was dependent on ICAM-1/fg interaction as well as RhoA activity. ICAM-1 was concentrated in focal adhesion plaques when tumor cells were allowed to adhere on immobilized fg, suggesting a role in cell migration. The addition of fg induced a 3- to 6-fold enhancement of bladder tumor cell migration through HUVEC monolayers. This process was inhibited by an anti-ICAM-1 antibody blocking fg binding, demonstrating that ICAM-1/fg interaction was involved in the extravasation process. Finally, immunohistological studies revealed that the expression of ICAM-1 was closely associated with an infiltrative histological phenotype.

Modelling of cell proliferation: nuclear versus membrane labelling.

Cell proliferation is a fundamental process involved in growth, development and oncogenesis. Monitoring and quantification of proliferation are essential to analyse the behaviour of cells drug-treated or not. Flow cytometry assessment of cell proliferation requires mathematical models to extract information of interest from fluorescence distributions. Various methods are available for cell cycle analysis, including estimation of cell phase durations and doubling time. In this context, we compare widely used flow cytometric methods based on nuclear labelling (using BrdUrd incorporation in combination with DNA content) to membrane labelling (using intercalating dyes PKH).

[Breast carcinoma with predominant neuroendocrine differentiation]. / Adénocarcinome mammaire avec composante neuroendocrine majoritaire.

Neuroendocrine differentiation can be identified in a subset of human breast carcinomas, either as scattered cells or as a predominant neuroendocrine component. We report a case of an invasive breast carcinoma largely composed of neuroendocrine cells. Eight years after a left mammary lumpectomy for a pT2N1MO SBR III invasive ductal carcinoma, a 67-years-old woman presented with a metastastic neuroendocrine sternal mass. To establish a relationship between mammary carcinoma and bone metastasis, histological slides of both the breast tumor and axillary lymph nodes were reviewed, and an immunohistochemical study was performed. They showed that: a) the mammary carcinoma was composed of a majority of small and large neuroendocrine cells synaptophysin +, NCAM+, chromogranin - (80%), associated with 2 other differentiated non endocrine components, one of metaplastic squamous carcinoma (10%) and the other of ductal carcinoma (10%); b) 4 axillary lymph nodes were involved by the ductal component which contained few NCAM + but synaptophysin - cells; c) Estrogen and progesterone receptors and HER2 were negative in the breast tumor and the metastatic nodes. We discuss the histogenesis of composite mammary carcinomas with neuroendocrine differentiation, the outcome of each component and the prognostic relevance of such a diagnosis.

Inhibition of c-Jun N-terminal kinase by SP600125: a cDNA microarray analysis.

BACKGROUND: In a previous investigation, we showed that the janus kinase (JNK) inhibitor SP600125 induced several phenotypic and genomic changes in leukemia cells. However, the molecular mechanisms that sustain these changes remain unknown. The purpose of the present study was to examine gene expression changes in THP-1 leukemia cells treated with SP600125. MATERIALS AND METHODS: Gene expression levels were investigated using Affymetrix hybridization technology and quantitative reverse transcriptase polymerase chain reaction. RESULTS: Affymetrix technology showed that the expression of 1,038 genes with a biological process description well known in gene ontology was modulated. Fifteen genes were related to kinases or phosphatases, 20 genes were involved in the cell cycle regulation, and 23 genes were involved in apoptosis. A network of 15 correlated genes was obtained showing a primordial role for the myelocytomatosis viral oncogene homolog (MYC). CONCLUSION: These findings show that SP600125 exhibits cytostatic and cytolytic activities through MYC gene modulation.

Significance of low levels of thyroglobulin in fine needle aspirates from cervical lymph nodes of patients with a history of differentiated thyroid cancer.

OBJECTIVE: Measurement of thyroglobulin in the washout of lymph node (LN) fine needle aspirates is recommended in the follow-up of patients with differentiated thyroid cancer (DTC). The significance of low fine needle aspirates thyroglobin (FNATg) levels remains a question, which we addressed. METHOD: Prospective study comparing FNATg with FNA cytology. Exploration of 34 DTC patients (53 cervical LNs), 26 non-thyroidectomized patients with a thyroid-unrelated cervical mass (negative controls) and 13 with 21 thyroid nodules (positive controls). The 12 DTC patients (19 LNs) with a malignant FNA cytology and/or high FNATg level received LN surgery (11 patients) or I(131)-iodine treatment (1 patient) and the outcome measure was pathological or scintigraphic evidence of DTC LN metastasis. RESULTS: All 26 negative controls showed FNATg <1 ng/FNA and all 21 positive controls showed high levels of FNATg (127-210,000 ng/FNA, median 38,000). Among DTC patients in 25 LNs with a benign FNA cytology, FNATg was undetectable in 24 and low in 1 (6 ng/FNA); in 19 LNs with a malignant FNA cytology, FNATg was high in 17 (80-140,000 ng/FNA, median 7174 ng/FNA) and low in 2 (6.6 and 7.1 ng/FNA), which proved to be low Tg immunostaining oncocytic DTC metastasis; in 9 LNs with a non-informative cytology, FNATg was undetectable in 8 but 11,825 ng/FNA in 1, which proved a DTC metastasis. Measurement of FNA albumin demonstrated that contamination of FNA by serum proteins was negligible. CONCLUSION: Low FNATg levels can indicate a DTC metastasis. It cannot be related to clinically relevant levels of serum Tg.

Sleep apnea is associated with bronchial inflammation and continuous positive airway pressure-induced airway hyperresponsiveness.

BACKGROUND: Obstructive sleep apnea syndrome (OSA) is associated with systemic and upper airway inflammation. Pharyngeal inflammation has a potential role in upper airway collapse, whereas systemic inflammation relates to cardiovascular morbidity. However, the presence of an inflammatory involvement of lower airway has been poorly investigated. OBJECTIVE: The aim of the study was to demonstrate an inflammatory process at the bronchial level in patients with OSA and to analyze effects of continuous positive airway pressure (CPAP) application and humidification on bronchial mucosa. METHODS: The study was conducted by using sequential induced sputum for cell analysis and IL-8 production, nitric oxide exhalation measurement, and methacholine challenge before and after CPAP. RESULTS: Bronchial neutrophilia and a high IL-8 concentration were observed in untreated OSA compared with controls (75% +/- 20% vs 43% +/- 12%, P < .05; and 25.02 +/- 9.43 ng/mL vs 8.6 +/- 3.7 ng/mL, P < .001, respectively). IL-8 in sputum supernatant was correlated to apnea hypopnea index (P < .01; r = 0.81). After 1 month of CPAP, this inflammatory pattern remained unchanged, and an increase in airway hyperresponsiveness (AHR) was observed (P < .001). CONCLUSION: Obstructive sleep apnea syndrome is associated with bronchial inflammation. Our data demonstrate CPAP effect on the development of AHR, possibly facilitated by the pre-existing inflammation. Both issues should be evaluated during long-term CPAP use. CLINICAL IMPLICATIONS: Results showing a spontaneous bronchial inflammation in OSA and the development of a CPAP-related AHR require a long-term follow-up to evaluate consequences on chronic bronchial obstruction.

In vitro tumoral progression of human bladder carcinoma: role for TGFbeta.

OBJECTIVE: Investigating whether extracellular factors are possible actors in tumoral progression in bladder carcinoma. METHODS: RT112/G2 bladder tumour cells were grown in presence of TGFbeta and analysed by immunological and cDNA microarray techniques. RESULTS: TGFbeta inhibited cell proliferation, reduced TNFalpha- and IFNgamma-induced apoptosis by decreasing TNFalpha-RI and IFNgamma-R antigen expression. It also inhibited cleaved caspase 8 and 9 expression, decreased E-cadherin, and increased BclxL and cyclooxygenase-2 expression. The cDNA microarray approach showed that TGFbeta up-regulated the expression of genes with defined roles in tumoral progression sometimes associated with poor outcome in bladder cancer. CONCLUSION: These results suggest that a part of the bladder tumoral progression process may be related to the action of exogenous TGFbeta confirming the possible role for the microenvironment.

Plasminogen- and colony-stimulating factor-1-associated markers in bladder carcinoma: diagnostic value of urokinase plasminogen activator receptor and plasminogen activator inhibitor type-2 using immunocytochemical analysis.

The expression of plasminogen- and colony-stimulating factor-1-associated markers was first investigated in seven bladder carcinoma cell lines and in 15 primary bladder tumors using RT-PCR (mRNAs), zymography (protein activity), ELISA and immunocytochemistry analysis (ICC) (protein levels). The mRNAs expression, the activity and the levels of the secreted proteins were not informative. Only urokinase plasminogen activator receptor (uPA-R/CD87) and possibly plasminogen activator inhibitor type-2 (PAI2) antigen expression at the cellular levels seem to be useful markers. uPA-R antigen expression correlated with the secretion of hepatocyte growth factor (HGF) ( P=0.016) and the motility of the bladder tumor cells ( P=0.014), two markers associated with a poor prognosis in bladder carcinoma. To validate our technique and confirm these preliminary results, uPA-R and PAI2 antigen expression was determined in the imprints from 129 resected bladder carcinoma fragments. uPA-R correlated with the grade ( P=0.002), tumor invasion ( P=0.003) and the ploidy ( P=0.05) of the bladder carcinomas and with the low overall survival ( P=0.045) of the patients. PAI2 correlated only with the stage ( P=0.02) and low overall survival ( P=0.038). We conclude that in bladder carcinomas, studying the transcripts of PAs, PAIs, CSF-1 and its receptor, as well as measuring their concentration or activity in culture supernatants was of no clinical interest in terms of diagnostic or prognostic value. Only the ICC of uPA-R, which correlated with the major histopathological parameters of tumors and the low overall survival, proved to be a diagnostic and prognostic marker.

Effects of Tumor Necrosis Factor-α (TNFα) and Interferon-γ (IFNγ) on Gene Expression Profiles in Bladder Carcinoma Cells Using Oligonucleotide Microarray Analysis.

BACKGROUND: TNFα and IFNγ, two main cytokines secreted in the urine of bladder cancer patients after Bacillus Calmette Guerin immunotherapy (BCG therapy), exert various responses ranging from growth arrest, apoptosis, phenotypic changes and differentiation. MATERIALS AND METHODS: To identify their transcriptional and translational targets, the highly sensitive bladder cancer cell line (RT112) was treated for 24 hours with increasing doses of IFNγ or TNFα and analyzed for cellular and molecular changes using a cDNA microarray technique (Transcriptome) containing 800 genes. RESULTS: High doses (>10 ng/ml) induced an apoptotic cell death, whereas low doses (<5 ng/ml) induced a survival program. TNFα-inducible genes, IFNγ-inducible genes and genes modulated by TNFα and IFNγ together were identified. All were related to the tumor progression program including cell proliferation, apoptosis/survival, angiogenesis and metastatic processes. CONCLUSION: These results suggest that the transcriptomic approach could be a good methodology to determine the molecular mechanisms involved in bladder tumor progression processes in relation to a low response to BCG treatment. However, mRNA and protein expression did not always correlate, suggesting that translational regulation is a vital process in bladder tumor progression.

Improved detection of urothelial carcinomas with fluorescence immunocytochemistry (uCyt+ assay) and urinary cytology: results of a French Prospective Multicenter Study.

The aim of the study was to assess the sensitivity and specificity of fluorescence immunocytochemistry (uCyt+ assay) as combined with urinary cytology for detection of primary and recurrent urothelial carcinomas. We analyzed 694 urinary samples from 236 new symptomatic patients and 458 patients followed after transurethral resection (TUR) for bladder tumor. Lesions suspicious for cancer at cystoscopy were sampled by biopsies or TUR. Sensitivity and specificity of tests were calculated using cystoscopy and histopathology, whether or not combined as gold standards. In new symptomatic patients, sensitivity of uCyt+ was 40%, 88.2%, and 76.7%, whereas that of urinary cytology was 30%, 70.6%, and 83.3%, respectively, in G1, G2, and G3 tumors. In follow-up cases, sensitivity of uCyt+ was 61.9%, 66.7%, and 76.9%, whereas that of urinary cytology was 38.1%, 58.3%, and 64.1%, respectively, in G1, G2, and G3 tumors. The combination of uCyt+ and urinary cytology significantly increased mean sensitivity in newly diagnosed cases (86.4% versus 71.2% with urinary cytology only, p < 0.05), as well as in patients followed after TUR (79.3% versus 55.2%, p < 0.001). Specificity of uCyt+ and urinary cytology was identical in new patients (83.3%) and was 81.9% and 86.2%, respectively, in patients followed after TUR. In patients with negative cystoscopy, positive uCyt+ tests had a strong predictive value for tumor recurrence at 1 year (47.0% versus 11.9% in patients with negative assay, p < 0.01). We conclude that combining uCyt+ with urinary cytology improves the detection of urothelial carcinomas as well in patients with symptoms suggesting bladder cancer as in those followed after treatment.

Accuracy of human papillomavirus testing in primary screening of cervical neoplasia: results from a multicenter study in India.

The knowledge that cervical neoplasia are caused by human papillomavirus (HPV) infection has led to the evaluation of its role in screening. We evaluated the accuracy of HPV testing by Hybrid capture II (HC II) method in detecting cervical intraepithelial neoplasia grade 2 and 3 (CIN 2 and 3) lesions in 4 cross-sectional studies with common protocol and questionnaire in 3 different locations (Kolkata, Mumbai and Trivandrum) in India. These studies involved 18,085 women aged 25-65 years. The reference standard for final diagnosis was a combination of colposcopy/biopsy. All women were investigated with colposcopy and 3,116 received directed biopsy. The sensitivity of HPV testing for detecting CIN 2-3 lesions varied from 45.7% to 80.9% across the study sites; the specificity varied from 91.7% to 94.6% and the positive predictive value from 6.7% to 13.7%. Retesting of 298 randomly chosen denatured samples in France revealed an agreement rate of 85.9% and a kappa-value of 0.72. Although HPV testing seems to be a promising approach for cervical cancer prevention, a large range in sensitivity was observed in our study, possibly due to variations in the quality of specimen collection and reference standards. A higher sensitivity was associated with the center performing the test well. Further developments in terms of more reproducible, less expensive and less sophisticated testing are essential to make the test feasible and effective in low-resource settings.