A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.     

PKR activity is required for acute leukemic cell maintenance and growth: A role for PKR‐mediated phosphatase activity to regulate GSK‐3 phosphorylation

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p‐T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant‐negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time‐dependent augmentation of AKT S473 and GSK‐3α S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK‐3α was not dependent on PI3K activity. PKR inhibition augmented levels of p‐S473 AKT and p‐S21/9 GSK‐3α/β in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK‐3α or β phosphorylation in the presence of the AKT inhibitor, A443654. Pre‐treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2α, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2α phosphorylation. The effects of PKR inhibition on AKT and GSK‐3 phosphorylation were found to be, in part, PP2A‐dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK‐3. J. Cell. Physiol. 221: 232–241, 2009. © 2009 Wiley‐Liss, Inc     

Downhill versus Barrier-Limited Folding of BBL: 3. Heterogeneity of the Native State of the BBL Peripheral Subunit Binding Domain and Its Implications for Folding Mechanisms

Protein folding studies are generally predicated on Anfinsen's dogma that there is a unique native state of a protein. However, this is not always the case. NMR measurements of BBL, for example, find a decrease in helicity of helix 2 surrounding His166 on its protonation, which, with other experimental data, suggests that the native state can occupy two or more conformations. Here, we analysed the native structure of BBL as a function of pH, temperature and ionic strength, along with a truncated BBL construct, by extensive all-atom molecular dynamics simulations in explicit solvent, corresponding to at least 400 ns of trajectories collected for each set of conditions. The native state was heterogeneous under a variety of conditions, consisting of two predominant conformations. This equilibrium changed with conditions: protonation of His166 at low pH shifted the equilibrium in favour of a less ordered conformer, while high ionic strength at neutral pH shifted the equilibrium to a more ordered conformer. Furthermore, high temperature and truncation of the sequence also shifted the equilibrium toward the less ordered conformer. Importantly, conformational heterogeneity in a native structure that changes with conditions will lead to deviations from the classic two-state behaviour during the barrier-limited unfolding of a protein. In particular, some regions of the protein will appear to unfold asynchronously and some residues will have anomalous thermal titration curves and unusual baseline behaviour monitored microscopically by NMR spectroscopy and macroscopically by calorimetry and other techniques. Such data could otherwise be interpreted as evidence for barrier-free downhill folding. Any biological significance of downhill folding of BBL appears to be ruled out by recent crystallographic studies on the reaction cycle of the BBL-equivalent domain in a pyruvate dehydrogenase multienzyme complex in which the domain remains of constant structure.     

New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib,dasatinib, and nilotinib in human plasma

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.     

Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

Background  

The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI).     

Carcinoma and multiple lymphomas in one patient: establishing the diagnoses and analyzing risk factors

Multiple malignancies may occur in the same patient, and a few reports describe cases with multiple hematologic and non-hematologic neoplasms. We report the case of a patient who showed the sequential occurrence of four different lymphoid neoplasms together with a squamous cell carcinoma of the lung. A 62-year-old man with adenopathy was admitted to the hospital, and lymph node biopsy was positive for low-grade follicular lymphoma. He achieved a partial remission with chemotherapy. Two years later, a PET-CT scan showed a left hilar mass in the lung; biopsy showed a squamous cell carcinoma. Simultaneously, he was diagnosed with diffuse large B cell lymphoma in a neck lymph node; after chemo- and radiotherapy, he achieved a complete response. A restaging PET-CT scan 2 years later revealed a retroperitoneal nodule, and biopsy again showed a low-grade follicular lymphoma, while a biopsy of a cutaneous scalp lesion showed a CD30-positive peripheral T cell lymphoma. After some months, a liver biopsy and a right cervical lymph node biopsy showed a CD30-positive peripheral T cell lymphoma consistent with anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. Flow cytometry and cytogenetic and molecular genetic analysis performed at diagnosis and during the patient’s follow-up confirmed the presence of two clonally distinct B cell lymphomas, while the two T cell neoplasms were confirmed to be clonally related. We discuss the relationship between multiple neoplasms occurring in the same patient and the various possible risk factors involved in their development.