ATM protein and p53-serine 15 phosphorylation in ataxia-telangiectasia (AT) patients and at heterozygotes

ATM (ataxia-telangiectasia mutated) gene plays a central role in the DNA-damage response pathway. We characterized the ATM protein expression in immortalized cells from AT and AT-variant patients, and heterozygotes and correlated it with two ATM-dependent radiation responses, G1 checkpoint arrest and p53-Ser 15 phosphorylation. On Western blots, the full-length ATM protein was detected in eight of 18 AT cases, albeit at 1–32% of the normal levels, whereas a truncated ATM protein was detected in a single case, despite the prevalence among cases of truncation mutations. Of two ataxia without telangiectasia [A-(T)] cases, one expressed 20% and the other ~70% of the normal ATM levels. Noteworthy, among ten asymptomatic heterozygous carriers for AT, normal amounts of ATM protein were found in one and reduced by 40–50% in the remaining cases. The radiation-induced phosphorylation of p53 protein at serine 15, largely mediated by ATM kinase, was defective in AT, A(-T) and in 2/4 heterozygous carriers, while the G1 cell cycle checkpoint was disrupted in all AT and A(-T) cases, and in 3/10 AT heterozygotes. Altogether, our study shows that AT and A(-T) cases bearing truncation mutations of the ATM gene can produce modest amounts of full-length (and only rarely truncated) ATM protein. However, this limited expression of ATM protein provides no benefit regarding the ATM-dependent responses related to G1 arrest and p53-ser15 phosphorylation. Our study additionally shows that the majority of AT heterozygotes express almost halved levels of ATM protein, sufficient in most cases to normally regulate the ATM-dependent DNA damage-response pathway. © 2000 Cancer Research Campaig

Androgen-regulated gastrin-releasing peptide receptor expression in androgen-dependent human prostate tumor xenografts

Human prostate cancer (PC) overexpresses the gastrin-releasing peptide receptor (GRPR). Radiolabeled GRPR-targeting analogs of bombesin (BN) have successfully been introduced as potential tracers for visualization and treatment of GRPR-overe

Pre-maturation with cAMP modulators in conjunction with EGF-like peptides during in vitro maturation enhances mouse oocyte developmental competence

Recent studies have independently shown that cyclic adenosine 3'5'-monophosphate (cAMP) modulation prior to in vitro maturation (IVM) and epidermal growth factor (EGF)-like peptide supplementation during IVM improve subsequent oocyte developmental outcomes. This study investigated the effects of an IVM system that incorporates these two concepts. Cumulus-oocyte complexes (COCs) were collected from pre-pubertal mice either 46 hr post-equine chorionic gonadotropin (eCG) (IVM) or post-eCG + post-human chorionic gonadotropin (hCG) stimulation (in vivo maturation; IVV). IVM COCs were treated with the cAMP modulators forskolin and IBMX for 1, 2, or 4 hr (pre-IVM phase) prior to IVM. COCs then underwent IVM with the EGF-like peptides amphiregulin or epiregulin, or with the common IVM stimulants follicle-stimulating hormone (FSH) or EGF. A pre-IVM phase increased the size of the subsequent blastocysts' inner-cell-mass compared to standard IVM, regardless of IVM treatment (P < 0.05). Unlike FSH or EGF, amphiregulin or epiregulin significantly increased blastocyst quality (trophectoderm and total cell numbers) and/or yield (P < 0.01) compared to standard IVM, and were the only treatments that produced blastocysts comparable to IVV-derived blastocysts. Forskolin acutely up-regulated EGF-like peptide mRNA expression after a 2-hr pre-IVM phase (P < 0.001), although EGF receptor and ERK1/2 activities were not significantly different than control. IVV-like levels of EGF-like peptide mRNA expression during IVM were maintained only by supplementing with EGF-like peptides and EGF, since expression levels induced by FSH were significantly lower in vitro than during IVV. However, EGF receptor and ERK1/2 phosphorylation levels were not significantly different across treatment groups. In conclusion, a pre-IVM phase in conjunction with IVM in the presence of EGF-like peptides endows high oocyte developmental competence, as evidenced by increased embryo yield and/or quality relative to FSH and EGF.D. Richani, X. Wang, H. Zeng, J.E.J. Smitz, J.G. Thompson, and R.B. Gilchris

Genetic Dissection of Epidermal Growth Factor Receptor Signaling during Luteinizing Hormone-Induced Oocyte Maturation

Recent evidence that luteinizing hormone (LH) stimulation of ovulatory follicles causes transactivation of the epidermal growth factor receptor (EGFR) has provided insights into the mechanisms of ovulation. However, the complete array of signals that promote oocyte reentry into the meiotic cell cycle in the follicle are still incompletely understood. To elucidate the signaling downstream of EGFR involved in oocyte maturation, we have investigated the LH responses in granulosa cells with targeted ablation of EGFR. Oocyte maturation and ovulation is disrupted when EGFR expression is progressively reduced. In granulosa cells from mice with either global or granulosa cell-specific disruption of EGFR signaling, LH-induced phosphorylation of MAPK3/1, p38MAPK, and connexin-43 is impaired. Although the LH-induced decrease in cGMP is EGFR-dependent in wild type follicles, LH still induces a decrease in cGMP in Egfrdelta/f Cyp19-Cre follicles. Thus compensatory mechanisms appear activated in the mutant. Spatial propagation of the LH signal in the follicle also is dependent on the EGF network, and likely is important for the control of signaling to the oocyte. Thus, multiple signals and redundant pathways contribute to regulating oocyte reentry into the cell cycle

LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse

Premature ovarian failure and female infertility are frequent side effects of anticancer therapies, owing to the extreme sensitivity of the ovarian reserve oocytes to the damaging effects of irradiation and chemotherapy on DNA. We report here a robust protective effect of luteinizing hormone (LH) on the primordial follicle pool of prepubertal ovaries against the cisplatin (Cs)-induced apoptosis. In vitro LH treatment of prepubertal ovarian fragments generated anti-apoptotic signals by a subset of ovarian somatic cells expressing LH receptor (LHR) through cAMP/PKA and Akt pathways. Such signals, reducing the oocyte level of pro-apoptotic TAp63 protein and favoring the repair of the Cs-damaged DNA in the oocytes, prevented their apoptosis. Noteworthy, in vivo administration to prepubertal female mice of a single dose of LH together with Cs inhibited the depletion of the primordial follicle reserve caused by the drug and preserved their fertility in reproductive age, preventing significant alteration in the number of pregnancy and of delivered pups. In conclusion, these findings establish a novel ovoprotective role for LH and further support the very attracting prospective to use physiological 'fertoprotective' approaches for preventing premature infertility and risks linked to precocious menopause in young patients who survived cancer after chemotherapy

Environmental impact of inhalers for respiratory diseases: decreasing the carbon footprint while preserving patient-tailored treatment

Patients with asthma and Chronic Obstructive Respiratory Disease (COPD) rely on three main device classes for inhalation therapy: metered-dose inhalers (MDIs), dry powder inhalers (DPIs) and soft-mist inhalers (SMIs). The carbon footprint (CF) of these inhalers differs with MDIs having a higher impact than DPIs and SMIs due to the propellant in MDIs. However, the certified CF of specific MDI products may differ significantly. MDIs still represent an essential option for many patients. Consequently, novel approaches shall be considered to balance environmental goals with patient health and well-being while maintaining a diverse range of choices for patients and physicians

Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance

TSH resistance is one of the causes of congenital hypothyroidism with thyroid gland in situ. We recently identified families with dominant transmission of partial TSH resistance due to heterozygous inactivating mutations in TSH receptor (TSHR) gene. Although we documented a poor routing of TSHR mutants to the cell membrane, the mechanism responsible for dominant inheritance of partial TSH resistance remained unexplained. We therefore co-transfected Cos-7 cells with wild-type TSHR and mutant receptors found in these patients. A variable impairment of cAMP response to bTSH stimulation was observed, suggesting that inactive TSHR mutants can exert a dominant negative effect on wild-type TSHR. We then generated chimeric constructs of wild-type or inactive TSHR mutants fused to different reporters. By fluorescence microscopy and immunoblotting, we documented an intracellular entrapment, mainly in the endoplasmic reticulum, and reduced maturation of wild-type TSHR in the presence of inactive TSHR mutants. Finally, fluorescence resonance energy transfer and co-immunoprecipitation experiments were performed to study the molecular interactions between wild-type and mutant TSHRs. The results are in agreement with the presence of oligomers formed by wild-type and mutant receptors in the endoplasmic reticulum. Such physical interaction represents the molecular basis for the dominant negative effect of inactive TSHR mutants. These findings provide an explanation for the dominant transmission of partial TSH resistance. This is the first report linking dominant negative mutations of a G protein-coupled receptor to an abnormal endocrine phenotype in heterozygous patients

Luteinizing Hormone-Dependent Activation of the Epidermal Growth Factor Network Is Essential for Ovulation

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility