Frédéric Troalen

Retinoblastoma protein represses transcription by recruiting a histone deacetylase

The retinoblastoma tumour-suppressor protein Rb inhibits cell proliferation by repressing a subset of genes that are controlled by the E2F family of transcription factors and which are involved in progression from the G1 to the S phase of the cell cycle. Rb, which is recruited to target promoters by E2F1 (ref. 3), represses transcription by masking the E2F1 transactivation domain and by inhibiting surrounding enhancer elements, an active repression that could be crucial for the proper control of progression through the cell cycle. Some transcriptional regulators act by acetylating or deacetylating the tails protruding from the core histones, thereby modulating the local structure of chromatin: for example, some transcriptional repressors function through the recruitment of histone deacetylases. We show here that the histone deacetylase HDAC1 physically interacts and cooperates with Rb. In HDAC1, the sequence involved is an LXCXE motif, similar to that used by viral transforming proteins to contact Rb. Our results strongly suggest that the Rb/HDAC1 complex is a key element in the control of cell proliferation and differentiation and that it is a likely target for transforming viruses.

Progression of medullary thyroid carcinoma: assessment with calcitonin and carcinoembryonic antigen doubling times

OBJECTIVE The progression of medullary thyroid cancer is difficult to assess with imaging modalities; we studied the interest of calcitonin and carcinoembryonic antigen (CEA) doubling times and of Ki-67 labeling and mitotic index (MI). PATIENTS AND METHODS Fifty-five consecutive medullary thyroid carcinoma (MTC) patients with elevated calcitonin levels underwent repeated imaging studies in order to assess tumor burden and progression status. We looked for relationships between tumor burden and levels of calcitonin and CEA and between progression status according to the response evaluation criteria in solid tumors (RECIST) and calcitonin and CEA doubling times, and Ki-67 labeling and MI. RESULTS The calcitonin and CEA levels were correlated with tumor burden. Ten patients with calcitonin levels below 816 pg/ml had no imaged tumor foci. Among the 45 patients with imaged tumor foci, 19 had stable disease and 26 had progressive disease, according to the RECIST. The calcitonin and CEA doubling times were strongly related to disease progression, with very few overlaps: 94% of patients with doubling times shorter than 25 months had progressive disease and 86% of patients with doubling times longer than 24 months had stable disease. Ki-67 labeling and MI were not significantly associated with disease progression. CONCLUSION For MTC patients, the doubling times of both calcitonin and CEA are efficient tools for assessing tumor progression.

Evaluation of protocols for purification of mouse monoclonal antibodies. Yield and purity in two-dimensional gel electrophoresis.

Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.

Survival and reproductive function of 52 women treated with surgery and bleomycin, etoposide, cisplatin (BEP) chemotherapy for ovarian yolk sac tumor

BACKGROUND Ovarian yolk sac tumor (YST) is a very rare malignancy arising in young women. Chemotherapy has dramatically improved the prognosis. Current treatment consists of surgery followed by bleomycin, etoposide, and cisplatin (BEP) chemotherapy. However, given the rarity of this tumor, ovarian YST-specific survival and outcome after such treatment are not precisely known. PATIENTS AND METHODS This report concerns prospectively recorded cases that were either treated at Institut Gustave Roussy (Villejuif, France) or referred there for advice about therapy. From 1990 to 2006, 52 patients underwent surgery followed by BEP chemotherapy. Data on patient characteristics, treatment, survival, and fertility outcome were analyzed to assess treatment efficacy and gonadal toxicity after achieving a complete remission. RESULTS Thirty-five patients had stage I/II tumors while 17 patients presented with stage III/IV disease. With a median follow-up of 68 months, the overall 5-year survival and disease-free survival rates were 94% and 90%, respectively. Forty-one women underwent fertility-sparing surgery. Pregnancy was achieved in 12 of 16 (75%) women who attempted conception. Overall, 19 pregnancies have been recorded. CONCLUSIONS BEP chemotherapy following fertility-sparing surgery is a very effective treatment of ovarian YSTs. Most of the patients who attempt conception after complete remission will have children.

Structural probing of human lutropin using antibodies raised against synthetic peptides constructed by classical and multiple antigen peptide system approaches

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.

Ultrasensitive serum thyroglobulin measurement is useful for the follow-up of patients treated with total thyroidectomy without radioactive iodine ablation

CONTEXT Thyroglobulin (Tg) measurement is a major tool for the follow-up of differentiated thyroid cancer (DTC) patients; however, in patients who do not undergo radioactive iodine (RAI) ablation, normal ultrasensitive Tg levels measured under levothyroxine treatment (usTg/l-T4) are not well defined. OBJECTIVE AND DESIGN This single-center retrospective study assessed usTg/l-T4 level in 86 consecutive patients treated with total thyroidectomy without RAI ablation for low-risk DTC (n=77) or for tumors of uncertain malignant potential (TUMP) (n=9). RESULTS DTCS were classified as PT1, PT2, and PT3 in 75, 1, and 1 case respectively and PN0, PN1, and PNX in 40, 6, and 31 respectively. following surgery, ten patients had TG antibodieS (TGAB). Among those without TGAB, the first USTG/L-T4 determination obtained at a mean time of 9 months after surgery was 0.1NG/ML in 62% of cases, 0.3NG/ML in 82% of cases, 1NG/ML in 91%, and 2NG/ML in 96% of cases. after a median follow-up of 2.5 years (range: 0.6-7.2 years), one patient had persistent disease with an usTg/l-T4 at 11 ng/ml and an abnormal neck ultrasonography (US) and two patients had usTg/l-T4 level >2 ng/ml (3.9 and 4.9 ng/ml) with a normal neck US. Within the first 2 years following total thyroidectomy without RAI ablation, usTg/l-T4 level is ≤2 ng/ml in 96% of the cases. CONCLUSION After total thyroidectomy, sensitive serum Tg/l-T4 level is ≤2 ng/ml in most patients and can be used for patient follow-up.

Self-association and Domains of Interactions of an Amphipathic Helix Peptide Inhibitor of HIV-1 Integrase Assessed by Analytical Ultracentrifugation and NMR Experiments in Trifluoroethanol/H2O Mixtures

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147–175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H2O mixtures. In pure water the helix content is weak but increases regularly up to 50–60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The NH temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four α-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147–175 segment in integrase.

Grafting peptides onto polystyrene microplates for ELISA

Three peptides corresponding respectively to two Epstein-Barr viral epitopes and to the c-erbB-2 oncogene product were synthesized with the aim of developing an immunoenzymatic assay. Preliminary experiments indicated that the efficiency of the assay was profoundly affected by the nature of the solid phase for each peptide. In order to optimize the assay the three peptides were covalently coupled to functionalized polystyrene microplates which were used to immobilize both haptens and nucleic acids in a previous study. The results obtained indicate that the use of the carboxylated surfaces permits the linking strategy to be adapted to each peptide. Moreover, high sensitivities (5 x 10(-10)-1 x 10(-13) M) were obtained using amounts of the peptides much lower than those used in the standard system.

Peptide mapping of intersubunit and receptor interactions of human choriogonadotropin

Seven peptides covering the entire sequence of human choriogonadotropin (hCG) alpha-subunit, eight peptides covering the hCG beta-subunit sequence and two peptides, one of human beta-lutropin and one of beta-thyrotropin were synthesized. We checked their ability to prevent reassociation between hCG alpha- and beta-subunits and between hCG and its receptor. Only the alpha 1-22, alpha 59-92 and beta 1-16 peptides inhibited the reassociation between the alpha- and beta-subunits of hCG with an ED50 of respectively 2 mM, 2 mM and 4 mM. Using porcine Leydig cells in primary culture, we showed that alpha 33-59, alpha 41-59 and beta 1-16 peptides decreased both the specific binding to the cell surface and the internalization of [125I]hCG and [125I]porcine LH with ED50 of 0.3, 0.1 and 0.5 mM, respectively. From these results, the following minimal area may be assigned, (i) to the alpha-beta interaction: alpha 5-16, alpha 52-72 (or alpha 59-70) and beta 8-16, and (ii) to the hormone-receptor association: alpha 41-45 and beta 8-16.

Characterization of a cleavage product in the human choriogonadotropin β-subunit

Abstract The various molecular forms of human chorionic gonadotropin present in a crude preparation of urine from pregnant women were analyzed by two-dimensional gel electrophoresis and immunoblotting with monoclonal antibodies directed to synthetic peptides corresponding to the carboxyl-terminal part of either the α or β-subunit. Under reducing conditions, immunoblotting with antibodies directed to the β-subunit revealed the presence of a low-molecular-weight material of 22 kDa. This molecular form had large heterogeneity, as analyzed by isoelectrofocusing; it was immunoreactive with antibodies directed to the 111–145 region. Using microsequencing techniques, we found that the fragment had a NH 2 terminal portion corresponding to the sequence of the β-subunit appearing from residue 48. Thus, the 22-kDa fragment comprises the 48–145 portion of the β-subunit and is probably a cleavage product of the native protein with intrachain nicking.