Distinct MDM2 and P14ARF expression and centrosome amplification in well-differentiated liposarcomas.

Well-differentiated liposarcomas (WDLs) are common soft-tissue tumors in adults. They are characterized by large marker chromosomes and/or ring chromosomes containing 12q-derived sequences in which MDM2 is consistently amplified. WDLs are subdivided into two subtypes according to their karyotype. Type D cells exhibit a near-diploid karyotype, with very few or no chromosome changes. Type H cells exhibit a near-tetraploid karyotype and many structural changes. Expression of P14ARF, MDM2, and TP53 proteins was assayed in the two WDL subtypes to establish whether distinct expression profiles correlated with cell ploidy. Although a transcriptionally functional TP53 was present in most tumors independent of their karyotype, type H cells were characterized by high levels of P14ARF and MDM2 proteins. Although amplified within similar chromosome markers in type D tumors, MDM2 did not appear to be overexpressed. In addition, it was present as a C-terminal truncated protein, indicative of alternatively spliced variants of MDM2 mRNA. As the existence of karyotypically distinct tumors could result from alterations of the mitotic machinery, we investigated the centrosome behavior in the two WDL subtypes. Centrosome amplification occurred in WDL tumors types H and D independent of their ploidy status. Moreover, no functional centrosome difference was found between the two tumor subtypes.

Downregulation of caspases and Fas ligand expression, and increased lifespan of neutrophils after transmigration across intestinal epithelium.

During inflammatory bowel diseases, commitment of extravased polymorphonuclear leucocytes (PMN) to apoptosis is required for the resolution of inflammation. To investigate the effect of transepithelial migration on PMN apoptotic rates, PMN transepithelial migration was reproduced in vitro using T84 intestinal monolayers. Transepithelial migration was found to delay neutrophil apoptosis, and this survival effect correlated with a downregulation of the surface expression of Fas ligand (FasL) and with a decrease in both procaspases-3, and -8 mRNA and procaspases-3, -6, -7 and -8 protein levels. Moreover, neutrophil survival and FasL shedding mediated by transepithelial migration were abrogated by a broad-spectrum metalloproteinase inhibitor, BB-94. Although Erk1/2 and p38 MAPK were activated in transmigrated PMN, inhibition of these MAP kinases did not impair transmigration-induced PMN survival. Taken together, our results show that trans-epithelial migration induces the downregulation of proapoptotic proteins expression in transmigrated PMN, which results in their increased lifespan.

Impact of the oxaliplatin-5 fluorouracil-folinic acid combination on respective intracellular determinants of drug activity.

The combination of 5-fluorouracil-folinic acid and oxaliplatin has led to a significant improvement of chemotherapy efficacy in advanced pretreated colorectal cancer. The objective of the present study was, considering the oxaplatin-5-fluorouracil-folinic acid combination, to examine the impact of one given drug on the cellular determinants of cytotoxic activity of the other drug. These cellular factors were analysed on the human colon cancer cell line WiDr in clinically relevant conditions of drug exposure ('De Gramont' schedule) with oxaliplatin-folinic acid during 2 h followed by 5-fluorouracil 48 h. The DNA binding of oxaliplatin was significantly reduced by the presence of 5-fluorouracil but this effect was time-dependent and after 50 h the platinum incorporated into DNA was identical in controls and in the drug combination. In the presence of oxaliplatin, there was less formation of FUH(2) which is the first catabolite produced in the cascade of 5-fluorouracil metabolic degradation. The effects of drugs on cell cycle were quite different from one drug to the other with oxaliplatin inducing a shift towards G(2) accumulation and 5-fluorouracil-folinic acid to a greater proportion of cells in G(1)-S. When oxaliplatin and 5-fluorouracil-folinic acid were combined the cell cycle effects were very similar to that of the 5-fluorouracil-folinic acid sequence alone. Oxaliplatin was able to reduce thymidylate synthase activity with a marked impact 28 h after the beginning of cell exposure to the drug. The 5-fluorouracil-folinic acid drug sequence led to a profound reduction in thymidylate synthase activity and this decrease was not markedly enhanced by the presence of oxaliplatin. Regarding apoptosis, changes in mitochondrial membrane permeability were observed in the presence of the tested drugs and the impact of 5-fluorouracil-folinic acid was greater than that of oxaliplatin. The addition of oxaliplatin did not amplify the action of 5-fluorouracil-folinic acid upon mitochondrial membrane permeability change. The presence of oxaliplatin itself did not modify the intracellular concentration of total reduced folates. The fact that oxaliplatin may reduce 5-fluorouracil catabolism could be central in explaining the supra-additive interaction between these drugs.

Ternary combination of irinotecan, fluorouracil-folinic acid and oxaliplatin: results on human colon cancer cell lines.

A marked antitumour efficacy is currently obtained by oxaliplatin (LOHP)-fluorouracil (FU)-folinic acid (FA) combination and by CPT11-FU-FA combination. Logically, the triple association LOHP, CPT11 and FUFA will be soon tested in cancer patients. The aim of the present study was to compare two schedules combining SN38 (the active metabolite of CPT11, irinotecan) with FU-FA and LOHP. The two schedules differed by the SN38 position. The relative contribution of each drug in the resulting global cytotoxicity was evaluated. Two human colon cancer cell lines were used (WIDR and SW620 both p53 mutated). LOHP plus FA were applied for 2 h, just before a 48 h FU exposure. The SN38 sequence was applied for 24 h, starting either 48 h before LOHP-FA (schedule A), or just after LOHP-FA exposure (schedule B). Cytotoxicity was assessed by the 3-(4,5-demethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) test and drug interactions were analysed according to the Chou and Talalay method, based on the computation of a combination index (CI). The SN38 position significantly induces a shift from additivity-antagonism when SN38 was applied after LOHP, towards additivity-synergism when SN38 was applied first (P = 0.03). The relative contribution (RC) of each drug in the overall cytotoxicity of the triple combination was defined as the drug concentration giving 50% cell lethality (IC(50)) of the double association without that drug divided by the IC(50)of the triple association. Whatever the SN38 position, the larger contribution was made by LOHP (median RC = 2.4) and the smaller by SN38 (median RC = 1.1). In addition, the contribution of FUFA was improved when SN38 was applied first (median RC = 2.2) as compared to the opposite schedule (median RC = 1.2). Results were in agreement between the two explored cell lines. The present data should be taken into account when establishing the rationale of future trials combining CPT11, LOHP and FU-FA.

Caspase inhibition protects from liver injury following ischemia and reperfusion in rats.

Normothermic ischemia and reperfusion of the liver results in microcirculatory failure followed by necrosis and cell death. Recently, another type of cell death, apoptosis or programmed cell death, was found to be activated during the early phase of reperfusion after liver ischemia. Caspases are cysteine proteinases specifically involved in the initiation and execution phases of apoptosis. The aim of this study was to demonstrate that inhibition of apoptosis by a specific inhibitor of caspases might protect the liver against ischemia/reperfusion injury. Rats were divided into three groups: group 1, control, PBS administration; group 2, Z-Asp-cmk (Z-Asp-2,6-dichlorobenzoyl-oxymethylketone) treatment; group 3, sham-operated control animals. Z-Asp-cmk (0.5 mg Z-Asp-cmk dissolved in 300 microl PBS solution containing 1% DMSO) was injected intravenously, 2 min prior to induction of 120 min ischemia. Survival rates were compared and serum activities of aspartate aminotransferases and alanine aminotransferases were assessed in the blood collected from the suprahepatic vena cava. Histology of the liver was assessed 6 h after the end of ischemia. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling method (TUNEL method) and by electrophoresis for analysis of DNA fragmentation. Caspase activity was determined by measuring hydrolysis of the CPP32-like substrate Ac-DEVD-pNA and absorption of paranitroaniline. Z-Asp-cmk treatment significantly increased 7-day survival (95%) compared with that in nontreated rats (30%, P < 0.001). Serum activities of aminotransferases and the extent of liver congestion and necrosis were significantly (P < 0.001) decreased after treatment with Z-Asp-cmk. TUNEL-positive cells were detected 3-6 h after reperfusion in the control group. In Z-Asp-cmk pretreated rats, a dramatic decrease in the number of TUNEL-positive cells was observed. Analysis of DNA fragmentation of freshly isolated hepatocytes confirmed these results. Caspase activity was increased 3-6 h after reperfusion in the control group, but significantly (P < 0.001) decreased after treatment with Z-Asp-cmk. These findings demonstrate that liver injury following ischemia and reperfusion can be prevented by inhibition of caspases. Caspase inhibitors may have important implications for therapy in liver disease and after liver transplantation.

Application of an original RT-PCR-ELISA multiplex assay for MDR1 and MRP, along with p53 determination in node-positive breast cancer patients.

The long-term prognostic value of tumoural MDR1 and MRP, along with p53 and other classical parameters, was analysed on 85 node-positive breast cancer patients receiving anthracycline-based adjuvant therapy. All patients underwent tumour resection plus irradiation and adjuvant chemotherapy (the majority receiving fluorouracil-epirubicin-cyclophosphamide). Median follow-up for the 54 alive patients was 7.8 years. Mean age was 53.7 years (range 28-79) and 54 patients were post-menopausal. MDR1 and MRP expression were quantified according to an original reverse transcription polymerase chain reaction multiplex assay with colourimetric enzyme-linked immunosorbent assay detection (beta2-microglobulin as control). P53 protein was analysed using an immunoluminometric assay (Sangtec). MDR1 expression varied within an 11-fold range (mean 94, median 83), MRP within a 45-fold range (mean 315, median 242) and p53 protein from the limit of detection (0.002 ng mg(-1)) up to 35.71 ng mg(-1) (mean 1.18, median 0.13 ng mg(-1)). P53 protein was significantly higher in oestrogen receptor (ER)-negative than in ER-positive tumours (P = 0.039). The higher the p53, the lower the MDR1 expression (P = 0.015, r= -0.27). P53 was not linked to progesterone receptor (PR) status, S phase fraction, or MRP Significantly greater MDR1 expression was observed in grade I tumours (P = 0.029). No relationship was observed between MDR1 and MRP. Neither MDR1 nor MRP was linked to ER or PR status. Unlike MDR1, MRP was correlated with the S phase: the greater the MRP, the lower the S phase (P = 0.006, r = -0.42). Univariate Cox analyses revealed that MDR1, MRP, p53 and S phase had no significant influence on progression-free or specific survival. A tendency suggested that the greater the p53, the shorter the progression-free survival (P = 0.076 as continuous and 0.069 as dichotomous).

[Immunohistochemical evaluation of the in vitro bromodeoxyuridine labeling index. 236 breast cancers studies by image analysis]. / Evaluation immunohistochimique de l'index de marquage après incorporation in vitro de bromodéoxyuridine. A propos de 236 cancers mammaires étudiés par analyse d'image.

We studied with computerized image analysis 236 breast cancer samplings after in vitro bromodeoxyuridine incorporation and immunohistochemical revelation. Labeling index values were compared with the usual prognostic factors and with the other studies in the literature. We established a positive correlation between labeling index and tumor size, histoprognostic grading, phase S and DNA index. A high labeling index was correlated with the absence of hormonal receptors but not correlated with the other prognostic factors. These results on tumor kinetics are similar to those obtained by flow cytometry and from other studies in the literature. However, this technic using optical microscopy allows for reliable selection of tumoral cells. Furthermore, the semi-automated image analysis provides an objective and reproducible evaluation of the labeling index.

A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis.

Apoptosisis activated during the early phase of reperfusion after liver ischemia and after liver transplantation in animals. However, the molecular basis of ischemia-induced cell death remains poorly understood. In this study we show that hepatocytes from ischemic liver lobes undergo apoptosis after reperfusion. In vivo pretreatment of rats with a specific inhibitor of caspases abrogates the apoptotic response in ischemic liver lobes. Inhibition of apoptosis can be accounted for by total inhibition of caspase activation as assessed in an enzymatic assay and by specific affinity labeling. Treatment with a caspase inhibitor fully protects rats from death induced by ischemia/reperfusion. These findings indicate that liver injury after ischemia/reperfusion can be prevented by inhibition of caspases. Thus, caspase inhibitors may have important therapeutic implications in liver ischemic diseases and after liver transplantation.

Combination of irinotecan (CPT11) and 5-fluorouracil with an analysis of cellular determinants of drug activity.

We evaluated the combination SN38 (7-ethyl-10-hydroxycamptothecin) -5fluorouracil (5FU) +/- folinic acid (FA) on six human colon cancer cell lines expressing spontaneous sensitivity to both drugs. Tumoral parameters potentially related to drug sensitivity were investigated: topoisomerase I (topo I) cleavable complexes formed with SN38, thymidylate synthase (TS) activity, folylpolyglutamate synthetase activity and dihydropyrimidine dehydrogenase activity. Drugs (SN38 and/or 5FU +/- FA) were applied for 72 hr, either sequentially or together. The concentration ratio between SN38 and 5FU was 100. Cytotoxicity (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide] test), DNA flow cytometry and isobologram analysis (Chou and Talatay) were performed. Based on 5FU IC50 values and isobologram analyses, the most cytotoxic schedule was SN38 followed by 5FU - FA, with high synergistic effects. Flow cytometry indicated that SN38 induced a more or less marked S-G2 block in all cell lines. Sensitivity to SN38, 5FU +/- FA, or combinations were not linked to the potential above-cited tumoral parameters. Interestingly, an inverse correlation was demonstrated between TS activity and topo I cleavable complexes (r2 = 0.78, P = 0.019). These data emphasize the critical importance of the irinotecan-5FU schedule and strongly support this association for the treatment of potentially 5FU-sensitive tumors.

Detection of rare circulating breast cancer cells by filtration cytometry and identification by DNA content: sensitivity in an experimental model.

Current methods of detecting micrometastases in breast cancer fail in a large proportion of patients. Therefore an improved method for detection of metastases in blood samples could be of great clinical interest both for prognosis and selection of patients for adjuvant systemic therapy. We have developed a new non-invasive method which associates immuno-magnetic separation and filtration cytometry. The sensitivity of our procedure was evaluated in a model system using a mixture from a human breast cancer cell line (MCF-7) and a normal human blood sample. The identification of tumoral cells was achieved by measuring DNA content in comparison with standard cells. The lowest concentration of MCF-7 detected was 1 tumoral cell in 500,000 white blood cells. In addition, filtration cytometry provides a visual control of nuclei permitting the elimination of all doubtful cases and an automatic count of tumoral cells directly per ml of blood, which may be an independent predictor of early relapse. This new method may avoid unnecessary axillary lymph node dissection in patients with negative nodes. Our procedure seems suitable for the detection of rare circulating cells in routine laboratory testing and could be used in other applications.

Cell cycle expression of p53 protein, c-Myc gene product and tyrosine-phosphorylation level determined by image analysis in human breast cancer cells.

OBJECTIVE: To investigate the cell cycle expression of p53 protein, c-myc gene product and tyrosine phosphorylation level in human breast cancer cells. STUDY DESIGN: Using a multifluorescence imaging procedure, the concentration per cell in different phases of the cell cycle can be evaluated by analyzing the bivariate contour plot of DNA content versus antigen concentration. RESULTS: Low fluorescence intensity was observed in the G0/G1 phase for the three markers. The analysis of individual cells demonstrated that approximately 10% of cells were negative. During the G1/S transition, the fluorescence intensity of the three antigens increased rapidly. However, after the mild S-phase, the increase of c-myc was more marked than the tyrosine phosphorylation level, whereas p53 protein remained stable, with a slight tendency to decrease. CONCLUSION: This study confirmed that the p53 protein and c-myc gene product could perform a regulatory function in G1/S transition and, consequently, may play an important role in malignant transformation. Like-wise, the variations of tyrosine kinase activity were linked to cellular progression throughout the cell cycle and could be a useful marker of alteration in the growth-factor signaling pathway. Thus, the multifluorescence imaging procedure may provide useful information on the mechanisms of the cell cycle and on malignant transformation.

Cell cycle expression of estrogen receptors determined by image analysis on human breast cancer cells in vitro and in vivo.

We have investigated, by image analysis, the cell cycle expression of estrogen receptors (ER) on MCF-7 cell line and on MCF-7 xenografts. The results demonstrate, in vitro as well as in vivo, an increase of ER concentration during the G0/G1-phase, followed by a decrease during the S-phase until the late S-phase where a rapid increase was noted. These results confirm that estrogens are involved in the DNA synthesis since ER is expressed in vivo at a maximal level in the late G1. In presence of saturating concentrations of 17 beta-estradiol, the mean ER concentration in G0/G1 phase is significantly decreased compared with the control cells cultured in estrogen-deprived medium. This indicates that 17 beta-estradiol down-regulates ER preferentially in the G0/G1 phase. These data suggest that ER in S and G2/M phases is unable to interact with its ligand. Consequently, estrogens may have no effects on the entry of cells in mitosis. Finally, after long-term tamoxifen treatment of MCF-7 xenografts, a tamoxifen-resistant tumor was developed which was characterized by a change in the profile of ER concentration during the G0/G1 phase. In conclusion, it is possible that the differences in cell cycle distribution of ER could be correlated with different phenotypes of breast cancer and also with different clinical phases of tumoral evolution. However, it remains to be known what is the clinical significance of the ER cell cycle expression in relation to tumor aggressiveness and survival.

Cell cycle expression of steroid receptors determined by image analysis on human breast cancer cell line: a hypothesis on the effects of antiestrogens.

Tamoxifen is the widespread anti-hormonal compound used for the treatment of human breast cancer. It is admitted that its effects are mediated via estrogen receptors (ER) but the molecular basis of its activity has yet to be clearly defined. In this work, we have developed a new image cytometry procedure in order to clarify the interactions between steroid receptors and tamoxifen at the cell cycle kinetic level. On untreated cells, an increase of ER level and a decrease of progesterone receptor (PR) level during the G0/G1 phase were demonstrated. Then, the ER and PR levels fell during the S-phase until the beginning of G2/M phase, where an increase was observed, especially for PR. These results suggest that ER is synthesized preferentially during the G0/G1 transition and PR during the S/G2 transition. After short-term tamoxifen treatment an augmentation of ER level was observed which was not dose-dependent, suggesting an increase in receptor translation rather than an augmentation of ER synthesis. PR level declined in the majority of the population leading to a selection of a subset of proliferating PR negative cells after treatment. These data demonstrate that the synthesis of steroid receptors is linked with the progression of cells through the cell cycle and indicate that tamoxifen blocks MCF-7 cells in G1 via its interactions with ER. Our multifluorescence imaging procedure appears to provide a rapid and quantitative approach which is useful for investigating alterations in steroid receptors after endocrine treatment.

Immunohistochemical determination of nuclear antigens by colour image analysis: application for labelling index, estrogen and progesterone receptor status in breast cancer.

The immunohistochemical evaluation of 5-bromo-2'-deoxyuridine (BrdU) labelling index, estrogen (ER) and progesterone receptor (PR) status was carried out on the automated computer-assisted image analysis station BIOCOM 500. Special software has been developed to measure nuclear antigens using the immunoperoxidase method with the Harris hematoxylin counterstain. The analysis was based on the different light adsorption spectra of the chromogen diaminobenzidine and Harris's hematoxylin coloration when exposed to light of differing wavelengths. The results obtained by image analysis were compared to previously validated methods. The thymidine labelling index performed by manual procedures and BrdU incorporation performed by image analysis were comparable (linear correlation coefficient r = 0.76, P < 0.001). Comparison of image analysis and dextran coated charcoal assay for ER and PR content revealed excellent sensitivities and specificities (linear correlation coefficient r = 0.87, P < 0.001 for ER and r = 0.93, P < 0.001 for PR). These data suggest that automated image analysis offers a reliable and reproducible procedure for measuring nuclear antigens.

Effects of tamoxifen on potential doubling time of human breast cancer cell line determined by image cytometry of double fluorescent BrdU and DNA labeling.

Tamoxifen is extensively used for the treatment of human breast cancer. However, the mechanisms by which antiestrogens regulate the growth of estrogen receptor positive tumors have not been totally defined. A new methodology, using automated image analysis BIOCOM 500, was developed for determining potential doubling time (Tpot) of tumors. This new method was checked on three different human breast cancer cell lines (MCF-7, CAL 85-1, CAL 148) in comparison with flow cytometry and then applied to determine the effects of short-term tamoxifen treatment on Tpot of MCF-7 cells. Using the resulting bivariate contour plot of blue fluorescence (DNA content) versus green fluorescence (Bromodeoxyuridine content), a labeling index (LI) value of 0.39 +/- 0.05 and a Tpot value of 21 +/- 2.09 hours were determined for MCF-7 cells. As expected, data demonstrated that 72 hours of 1 microM tamoxifen treatment decreased the LI to 35% by increasing the proportion of G0/G1 cells. It increased the Tpot to 35% compared to untreated cells (Tpot = 31.8 + 4 hours) by a lengthening of G0/G1 phase without changing the length of S phase (Ts = 10.2 +/- 1 hours). At suprapharmacological concentrations (5, 10 microM), an approximately 50% increase in Tpot was observed without modification in Ts. These data suggested a specific cell cycle action of tamoxifen which was probably mediated by mechanisms other than estrogen inhibition, since these experiments were performed in estrogen-deprived medium. In addition, the automated imaging procedure appears to provide a rapid and quantitative approach to determine Tpot in fine needle biopsies which is useful for investigating alterations in cell growth after endocrine treatment or chemotherapy.