Apoptosis and meiotic segregation in ejaculated sperm from Robertsonian translocation carrier patients.

BACKGROUND: To better understand the infertility of patients with Robertsonian translocation, the biochemical and ultrastructural apoptotic characteristics of apoptosis in the sperm of patients and fertile donors were studied. METHODS: Ejaculated sperm samples of seven Robertsonian translocation carriers and seven fertile donors were analyzed after cryopreservation. The proportion of both viable and dead spermatozoa expressing activated caspases was detected by flow cytometry through the use of different specific carboxyfluorescein-labeled caspase inhibitors. Sperm DNA fragmentation was evaluated by the TUNEL method. The percentages of intact spermatozoa or spermatozoa with ultrastructural features of apoptosis, immaturity or necrosis were estimated by electron microscopy. Meiotic segregation analysis was performed by FISH. RESULTS: Significantly lower concentration, forward motility and normal morphology of spermatozoa were found in ejaculated samples of the Robertsonian patients than fertile donors. Compared with the control group, in Robertsonian translocation carriers: (i) the caspase assays showed a significantly increased (P < 0.05) proportion of viable spermatozoa with activated poly-caspases (57.4 versus 25.8%), caspase-3 (43.5 versus 13.4%), caspase-8 (44.4 versus 17.1%) and caspase-9 (42.4 versus 10.0%); (ii) the rate of DNA fragmentation was higher (26.3 versus 12.8%); and (iii) sperm ultrastructural examination highlighted a higher percentage of immature (28.0 versus 10.0%) and apoptotic (24.5 versus 18.5%) spermatozoa. FISH study showed predominant normal/balanced spermatozoa (78.34-85.53%). CONCLUSIONS: These results show a predominant proportion of balanced and normal gametes and higher numbers of spermatozoa showing apoptosis and immaturity features in oligoasthenozoospermic Robertsonian translocation carriers than in fertile donors. This suggests defects in spermatogenesis and especially spermiogenesis of these infertile patients.

Fluorescence microscopy and flow cytometry in measuring activated caspases in human spermatozoa.

Staining of spermatozoa with the fluorescein-Val-Ala-Asp-fluoromethylketone has already been performed on ejaculated sperm samples, using fluorescence microscopy (FM) or flow cytometry (FCM) in order to score activated caspases. This assay may help in assessing apoptosis and its role in male fertility. The present study compares the above two techniques in order to adopt the most accurate method for detection in human frozen-thawed testicular, epididymal and ejaculated spermatozoa. The analyses were carried out on frozen/thawed testicular (n = 14), epididymal (n = 8) and ejaculated (n = 10) sperm samples. Activated caspases were detected in living spermatozoa using fluorescein-labelled inhibitors of poly-caspases (FLICA). For the measurements by FM, the same-observer and different-observer reliability were assessed in testicular and epididymal sperm samples. The inter-method (FM and FCM) reliability was assessed both in epididymal and ejaculated sperm samples. The reliability was evaluated by intraclass correlation coefficient (ICC) and the differences between paired measurements from the same sample were tested by Wilcoxon test for matched pairs. For the same-observer and the different-observer data, the ICC were 0.980 and 0.986. In testicular suspensions, the presence of different types of germinal and somatic cells hampers the differentiation of stained spermatozoa by FCM. For the inter-method reliability, the ICC was 0.903. A lower proportion of the viable spermatozoa stained with FLICA was observed by using FM (-6.60 +/- 7.38 %, mean +/- SD; p = 0.003) compared with FCM. To measure the proportion of spermatozoa with activated caspases by this test, FM is a highly accurate and reliable method whatever the sperm origin (ejaculate, epididymis, and testis). FCM cannot be used for testicular samples but seems to be more appropriate for analysis of epididymal and ejaculated sperm samples. The systematic lower proportion by FM in measuring the proportion of stained viable spermatozoa with FLICA involves that only the data measured by the same method (FM or FCM) may be compared.

Alkylation of prohibitin by cyclohexylphenyl-chloroethyl urea on an aspartyl residue is associated with cell cycle G(1) arrest in B16 cells.

BACKGROUND AND PURPOSE: Phenyl-chloroethyl ureas (CEUs) are a class of anticancer drugs that mainly react with proteins. Two molecules of this family, cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU) induced G(1)/S and G(2)/M cell cycle blocks, respectively. We hypothesised that these observations were linked to a differential protein alkylation pattern. EXPERIMENTAL APPROACH: Proteins from B16 cells incubated with [(14)C-urea]-CCEU and [(125)I]-ICEU were compared by 2D-analyses followed by MALDI-TOF identification of modified proteins and characterisation of the CCEU binding. Protein expression was investigated by Western blot analyses and cell cycle data were obtained by flow cytometry. KEY RESULTS: Several proteins (PDIA1, PDIA3, PDIA6, TRX, VDAC2) were alkylated by both ICEU and CCEU but beta-tubulin and prohibitin (PHB) were specifically alkylated by either ICEU or CCEU respectively. Specific alkylation of these two proteins might explain the observed difference in B16 cell cycle arrest in G(2) and G(1) phases respectively. Mass spectrometry studies on the alkylated prohibitin localised the modified peptide and identified Asp-40 as the target for CCEU. This alkylation induced an increased cellular content of PHB that should contribute to the accumulation of cells in G(1) phase. CONCLUSIONS AND IMPLICATIONS: This study reinforces our findings that CEUs alkylate proteins through an ester linkage with an acidic amino acid and shows that PHB alkylation contributes to G(1)/S arrest in CCEU treated B16 cells. Modification of PHB status and/or activity is an open route for new cancer therapeutics.

Hematological recovery and peripheral blood progenitor cell mobilization after induction chemotherapy and GM-CSF plus G-CSF in breast cancer.

In order to determine the effect of GM-CSF plus G-CSF in combination in breast cancer patients receiving an effective induction regimen, we compared hematological recovery and peripheral blood progenitor cell (PBPC) mobilization according to colony-stimulating factor (CSF) support. Forty-three breast cancer patients were treated by TNCF (THP-doxorubicin, vinorelbine, cyclophosphamide, fluorouracil, D1 to D4) with CSF support: 11 patients received GM-CSF (D5 to D14); 16 patients G-CSF (D5 to D14) and 16 patients GM-CSF (D5-D14) plus G-CSF (D10-D14). Between two subsequent cycles, progenitor cells were assessed daily, from D13 to D17. The WBC count was similar for patients receiving G-CSF alone or GM-CSF plus G-CSF, but significantly greater than that of patients receiving GM-CSF alone (P<0.001). The GM-CSF plus G-CSF combination led to better PBPC mobilization, with significantly different kinetics (P<0.001) and optimal mean values of CFU-GM, CD34+ cells and cells in cycle, at D15 compared to those obtained with G-CSF or GM-CSF alone. The significantly greater PBPC mobilization obtained with a CSF combination by D15 could be of value for PBPC collection and therapeutic reinjection after high-dose chemotherapies.

New method for stem cell quantification: applications to the management of peripheral blood stem cell transplantation.

A dramatic increase in peripheral blood stem cells (PBSC) is observed after high-dose chemotherapy followed by haematopoietic growth factors. The degree of mobilisation of PBSC is quantified by the level of clonogenic cells detected by CFU assays (CFU-GM or CFU-GEMM) or CD34+ cell determination. Working under the hypothesis that, in peripheral blood, mononuclear cells in DNA synthesis (MCDS) are proliferating stem cells, we decided to detect these cells by flow cytometric measurement of their DNA content. The relations between the number of MCDS and well-known haematopoietic progenitor indicators such as CFU-GM or CD34+ cells were analysed. We studied the kinetics of recruitment of PBSC in cancer patients, treated with rmeHuG-CSF following VP-16 cytoxan chemotherapy, until the first day of leukapheresis. For the 31 patients studied the individual curves of peripheral MCDS and CFU-GM reconstitutions showed identical profiles and a good correlation was noted between the numbers of peripheral MCDS and CFU-GM (r = 0.73). In the leukapheresis product, the predictive value of MCDS was equivalent to CFU-GM for PBSC quantification (r = 0.70). In conclusion, MCDS analysis by flow cytometry provides reliable results and appears to be an alternative to CFU-GM assay or CD34+ cell determination for PBSC quantification.

Mobilization of peripheral blood progenitor cells after induction chemotherapy (THP-doxorubicin-vinorelbine-cyclophosphamide-fluorouracil) and granulocyte colony-stimulating factor in breast cancer.

In order to evaluate the mobilization of peripheral blood progenitor cells (PBPC) after an effective induction regimen in breast cancer, we performed a study on 15 breast cancer patients. Between January 1995 and June 1996, these patients received TNCF (THP-doxorubicin. vinorelbine, cyclophosphamide, fluorouracil for four days, every 21 days) with G-CSF support (5 microg/kg for 10 days after chemotherapy) to reduce aplasia. This regimen is known to result in a complete pathological response in 30% of patients. Between two cycles of TNCF treatment, hematological recovery was observed. Progenitor cells (CFU-GM and CD34+ cells) and mononuclear cells in DNA synthesis (MCDS) counts were performed daily, between the 12th and 17th post-chemotherapy days (81 samples). The results showed a similarity for hematological recovery and PBPC mobilization kinetics depending on the number of treatment cycles. The three methods used for PBPC evaluation were well correlated (P < 0.01) with an optimal mean PBPC recruitment by the last day of G-CSF administration: respectively, 11 520 (1729-26539) CFU-GM/ml of blood, 249 (14-1160) CD34+ cells/microl of blood and 211 (21-554) MCDS/microl of blood. These results suggested that a daily injection of G-CSF after one or two TNCF cycles will produce an effective PBPC mobilization in comparison with currently used regimens.

rhGM-CSF vs placebo following rhGM-CSF-mobilized PBPC transplantation: a phase III double-blind randomized trial.

In this placebo-controlled randomized trial we evaluated the hematological and clinical effects of r-Hu GM-CSF after high-dose chemotherapy (HDC) followed by GM-CSF-mobilized PBPC transplantation. Fifty patients with poor prognosis malignancies were randomized in a double-blind study to receive either GM-CSF or placebo after HDC followed by PBPC rescue. For all patients, PBPCs were recruited using a combination of VP-16 (300 mg/m2 on days 1 and 2), cytoxan (3 g/m2 on days 3 and 4) and GM-CSF (5 micrograms/kg from day 5). No differences were demonstrated between the two groups in median time to neutrophil or platelet recoveries. There was no significant difference between the GM-CSF group and the placebo group in the median duration of post-transplant hospitalization, in the number of days of antibiotic treatment, in the number of infections and in red blood cell or platelet transfusion requirements. There was a significant difference with an advantage for the placebo group in the mean duration of febrile days (P = 0.01). We conclude that the administration of GM-CSF in patients transplanted with GM-CSF-mobilized PBPC is not associated with a clinical benefit in term of tempo of engraftment, numbers of documented infections, transfusion requirements and mucositis grading.

In addition to membrane injury, an affinity for melanin might be involved in the high sensitivity of human melanoma cells to hederacolchiside A1.

We previously reported that hederacolchiside A1 (Hcol A1), a new oleanene saponin isolated from Hedera colchica Koch (Araliaceae) exhibits a preferential cytotoxicity on a pigmented melanoma cell line. The present study confirms the high susceptibility of melanoma cell lines to this drug and shows concentrations producing a 50% decrease in cell content (IC50 values) inversely proportional to the melanin content of each cell line. At cytotoxic concentrations, Hcol A1 induces membrane-damaging effects within 6 h, cytoplasmic vacuolization within 24 h, and non-apoptotic cell death within 48 h. Using a new high-resolution magic-angle spinning nuclear magnetic resonance method, we have shown for the first time that this hederasaponin specifically interacts with melanin. The dissociation constant (2.7 mM) is comparable to those observed with drugs known to interact with melanin. Taking into consideration that the IC50 values were inversely proportional to the melanin in each cell line, our data suggest that, in addition to the delayed membrane injury induced by this drug, the ability of Hcol A1 to bind melanin could contribute to the higher toxicity of Hcol A1 in pigmented melanoma cells.

G2 accumulation and melanin overproduction in malignant melanocytes treated with a new nitrosourea.

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea), a new anticancer chloroethylnitrosourea (CENU) is being tested in a phase II clinical trial of disseminated melanoma. The antitumour effect of this drug is mainly due to DNA damage in malignant melanocytes. Recently, we have shown that this damage can induce apoptosis in some melanoma cell lines. In others, apoptosis is not clearly observed, although there is a strong cytostatic effect. In this paper, we have characterized the cytological effect of cystemustine on murine malignant melanocytes (B16 cell line) which are resistant to apoptosis induced by this CENU. The results show that 3 days after cystemustine treatment, these melanocytes had accumulated in phase G2 of the cell cycle. There was then a strong morphological modification during a long cytostatic phase up to 30 days after treatment. During this cytostatic phase, there was uncontrolled DNA synthesis and marked swelling. Also, tyrosinase activity, melanin content and the number of mature melanosomes were greatly increased. These results suggest that when malignant melanocytes are not able to undergo apoptosis after treatment with CENU, they accumulate in G2 and this is followed by enhancement of melanogenesis.

Association of N-acetyllactosamine with tumor progression in human breast cancer: a study using a 16 kDa chick embryo lectin.

This study documents modifications in the expression and the cellular distribution of binding sites for a 16 kDa chick embryo lectin (CL16-BS) in breast cancer. This lectin binds preferentially to terminal and penultimate N-acetyllactosamine residues (Galbeta1-4GlcNAc). BS density and distribution, studied by lectin binding followed by indirect immunofluorescence, were compared in normal breast tissues and 45 invasive carcinomas (lobular and ductal). Increased number of fluorescent epithelial cells (ETC+) were observed in normal ducts adjacent to lobular carcinomas and in tumors from both types when compared to normal glands. In ductal carcinomas, a significant diminution of ETC+ percentage was observed in the highest anatomopathological SBR grades: 32.7% in grade III, 80.8% in grade II and 66.5% in grade I (p<0.001). For both lobular and ductal carcinomas, ETC+ percentages were also positively correlated with low versus high MSBR grades (p<0.002). The subcellular distribution of CL16-BS varied according to the tumor type and/or the histological grades. It was mostly membrane-associated in low SBR and MSBR grades (p<0.001 and p<0.01, respectively) and cytoplasm-associated in high grades (p<0.02 and p<0.05). Some of these parameters were also correlated with certain other clinicopathological factors, such as tumor size (p<0.02), high S-phase cell fraction (p<0.04 and p<0.03) and low density estrogen receptors (p<0.05). Diminution in CL16-BS density and cytoplasmic versus membrane localization may be considered as indicators of tumor progression but not of metastasis.

Liver X Receptor activation downregulates AKT survival signaling in lipid rafts and induces apoptosis of prostate cancer cells.

Cholesterol is a structural component of lipid rafts within the plasma membrane. These domains, used as platforms for various signaling molecules, regulate cellular processes including cell survival. Cholesterol contents are tightly correlated with the structure and function of lipid rafts. Liver X receptors (LXRs) have a central role in the regulation of cholesterol homeostasis within the cell. Therefore, we investigated whether these nuclear receptors could modulate lipid raft signaling and consequently alter prostate cancer (PCa) cell survival. Treatment with the synthetic LXR agonist T0901317 downregulated the AKT survival pathway and thus induced apoptosis of LNCaP PCa cells in both xenografted nude mice and cell culture. The decrease in tumor cholesterol content resulted from the upregulation of ABCG1 and the subsequent increase in reverse cholesterol transport. RNA interference experiments showed that these effects were mediated by LXRs. Atomic force microscopy scanning of the inner plasma membrane sheet showed smaller and thinner lipid rafts after LXR stimulation, associated with the downregulation of AKT phosphorylation in these lipid rafts. Replenishment of cell membranes with exogenous cholesterol antagonized these effects, showing that cholesterol is a key modulator in this process. Altogether, pharmacological modulation of LXR activity could thus reduce prostate tumor growth by enhancing apoptosis in a lipid raft-dependent manner.

N-(4-iodophenyl)-N'-(2-chloroethyl)urea as a microtubule disrupter: in vitro and in vivo profiling of antitumoral activity on CT-26 murine colon carcinoma cell line cultured and grafted to mice.

The antitumoral profile of the microtubule disrupter N-(4-iodophenyl)-N'-(2-chloroethyl)urea (ICEU) was characterised in vitro and in vivo using the CT-26 colon carcinoma cell line, on the basis of the drug uptake by the cells, the modifications of cell cycle, and beta-tubulin and lipid membrane profiles. N-(4-iodophenyl)-N'-(2-chloroethyl)urea exhibited a rapid and dose-dependent uptake by CT-26 cells suggesting its passive diffusion through the membranes. Intraperitoneally injected ICEU biodistributed into the grafted CT-26 tumour, resulting thus in a significant tumour growth inhibition (TGI). N-(4-iodophenyl)-N'-(2-chloroethyl)urea was also observed to accumulate within colon tissue. Tumour growth inhibition was associated with a slight increase in the number of G2 tetraploid tumour cells in vivo, whereas G2 blockage was more obvious in vitro. The phenotype of beta-tubulin alkylation that was clearly demonstrated in vitro was undetectable in vivo. Nuclear magnetic resonance analysis showed that cells blocked in G2 phase underwent apoptosis, as confirmed by an increase in the methylene group resonance of mobile lipids, parallel to sub-G1 accumulation of the cells. In vivo, a decrease of the signals of both the phospholipid precursors and the products of membrane degradation occurred concomitantly with TGI. This multi-analysis established, at least partly, the ICEU activity profile, in vitro and in vivo, providing additional data in favour of ICEU as a tubulin-interacting drug accumulating within the intestinal tract. This may provide a starting point for researches for future efficacious tubulin-interacting drugs for the treatment of colorectal cancers.

Resveratrol increases BRCA1 and BRCA2 mRNA expression in breast tumour cell lines.

The phytochemical resveratrol, found in grapes, berries and peanuts, has been found to possess cancer chemopreventive effects by inhibiting diverse cellular events associated with tumour initiation, promotion and progression. Resveratrol is also a phyto-oestrogen, binds to and activates oestrogen receptors that regulate the transcription of oestrogen-responsive target genes such as the breast cancer susceptibility genes BRCA1 and BRCA2. We investigated the effects of resveratrol on BRCA1 and BRCA2 expression in human breast cancer cell lines (MCF7, HBL 100 and MDA-MB 231) using quantitative real-time RT-PCR, and by perfusion chromatography of the proteins. All cell lines were treated with 30 microM resveratrol. The expressions of BRCA1 and BRCA2 mRNAs were increased although no change in the expression of the proteins were found. These data indicate that resveratrol at 30 micro M can increase expression of genes involved in the aggressiveness of human breast tumour cell lines.

Isolation, purification and quantification of BRCA1 protein from tumour cells by affinity perfusion chromatography.

A new procedure for the isolation, purification and quantification of the product of the oncosuppressor gene brca1 in breast tissues, was carried out. It involves internal cell protein [35S]methionine labelling followed by two perfusion chromatographies. The first one is heparin affinity chromatography, to purify all of the cell DNA-binding proteins. A subsequent specific immunoprecipitation of BRCA1 protein was performed with an antibody raised against BRCA1. The immune complex was isolated using the second chromatographic step, Protein A affinity chromatography. The amount of BRCA1 expressed by cells was expressed as a ratio, in percent, calculated as follows: 100x amount of labelled DNA-binding proteins (dpm) that bound specifically to the anti-BRCA1 polyclonal antibodies (K-18)/amount of whole labelled DNA-binding protein (dpm) purified on a heparin column. Applications to MCF7 and T-47D human breast tumour cell lines, which were treated or not using 2 mM sodium butyrate demonstrated an increase in BRCA1 protein expression.