Early in vitro selection of eutypine-tolerant plantlets. Application to screening of Vitis vinifera cv. Ugni lanc somaclones

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Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells

Cisplatin produces good responses in solid tumours including small cell lung cancer (SCLC) but this is limited by the development of resistance. Oxaliplatin is reported to show activity against some cisplatin-resistant cancers but there is little known about oxaliplatin in SCLC and there are no reports of oxaliplatin resistant SCLC cell lines. Studies of drug resistance mainly focus on the cellular resistance mechanisms rather than how the cells develop resistance. This study examines the development of cisplatin and oxaliplatin resistance in H69 human SCLC cells in response to repeated treatment with clinically relevant doses of cisplatin or oxaliplatin for either 4 days or 2h. Treatments with 200ng/ml cisplatin or 400ng/ml oxaliplatin for 4 days produced sublines (H69CIS200 and H69OX400 respectively) that showed low level (approximately 2-fold) resistance after 8 treatments. Treatments with 1000ng/ml cisplatin or 2000ng/ml oxaliplatin for 2h also produced sublines, however these were not stably resistant suggesting shorter treatment pulses of drug may be more effective. Cells survived the first five treatments without any increase in resistance, by arresting their growth for a period and then regrowing. The period of growth arrest was reduced after the sixth treatment and the H69CIS200 and H69OX400 sublines showed a reduced growth arrest in response to cisplatin and oxaliplatin treatment suggesting that "regrowth resistance" initially protected against drug treatment and this was further upregulated and became part of the resistance phenotype of these sublines. Oxaliplatin dose escalation produced more surviving sublines than cisplatin dose escalation but neither set of sublines were associated with increased resistance as determined by 5-day cytotoxicity assays, also suggesting the involvement of regrowth resistance. The resistant sublines showed no change in platinum accumulation or glutathione levels even though the H69OX400 subline was more sensitive to buthionine sulfoximine treatment. The H69CIS200 cells were cross-resistant to oxaliplatin demonstrating that oxaliplatin does not have activity against low level cisplatin resistance. Relative to the H69 cells, the H69CIS200 and H69OX400 sublines were more sensitive to paclitaxel and taxotere suggests the taxanes may be useful in the treatment of platinum resistant SCLC. These novel cellular models of cisplatin and oxaliplatin resistant SCLC will be useful in developing strategies to treat platinum-resistant SCLC

Boolean Dynamics with Random Couplings

This paper reviews a class of generic dissipative dynamical systems called N-K models. In these models, the dynamics of N elements, defined as Boolean variables, develop step by step, clocked by a discrete time variable. Each of the N Boolean elements at a given time is given a value which depends upon K elements in the previous time step. We review the work of many authors on the behavior of the models, looking particularly at the structure and lengths of their cycles, the sizes of their basins of attraction, and the flow of information through the systems. In the limit of infinite N, there is a phase transition between a chaotic and an ordered phase, with a critical phase in between. We argue that the behavior of this system depends significantly on the topology of the network connections. If the elements are placed upon a lattice with dimension d, the system shows correlations related to the standard percolation or directed percolation phase transition on such a lattice. On the other hand, a very different behavior is seen in the Kauffman net in which all spins are equally likely to be coupled to a given spin. In this situation, coupling loops are mostly suppressed, and the behavior of the system is much more like that of a mean field theory. We also describe possible applications of the models to, for example, genetic networks, cell differentiation, evolution, democracy in social systems and neural networks.Comment: 69 pages, 16 figures, Submitted to Springer Applied Mathematical Sciences Serie

The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells

<p>Abstract</p> <p>Background</p> <p>Heparin affin regulatory peptide (HARP), also called pleiotrophin, is a heparin-binding, secreted factor that is overexpressed in several tumours and associated to tumour growth, angiogenesis and metastasis. The C-terminus part of HARP composed of amino acids 111 to 136 is particularly involved in its biological activities and we previously established that a synthetic peptide composed of the same amino acids (P111-136) was capable of inhibiting the biological activities of HARP. Here we evaluate the ability of P111-136 to inhibit <it>in vitro </it>and <it>in vivo </it>the growth of a human tumour cell line PC-3 which possess an HARP autocrine loop.</p> <p>Methods</p> <p>A total lysate of PC-3 cells was incubated with biotinylated P111-136 and pulled down for the presence of the HARP receptors in Western blot. <it>In vitro</it>, the P111-136 effect on HARP autocrine loop in PC-3 cells was determined by colony formation in soft agar. <it>In vivo</it>, PC-3 cells were inoculated in the flank of athymic nude mice. Animals were treated with P111-136 (5 mg/kg/day) for 25 days. Tumour volume was evaluated during the treatment. After the animal sacrifice, the tumour apoptosis and associated angiogenesis were evaluated by immunohistochemistry. <it>In vivo </it>anti-angiogenic effect was confirmed using a mouse Matrigel™ plug assay.</p> <p>Results</p> <p>Using pull down experiments, we identified the HARP receptors RPTPβ/ζ, ALK and nucleolin as P111-136 binding proteins. <it>In vitro</it>, P111-136 inhibits dose-dependently PC-3 cell colony formation. Treatment with P111-136 inhibits significantly the PC-3 tumour growth in the xenograft model as well as tumour angiogenesis. The angiostatic effect of P111-136 on HARP was also confirmed using an <it>in vivo </it>Matrigel™ plug assay in mice</p> <p>Conclusions</p> <p>Our results demonstrate that P111-136 strongly inhibits the mitogenic effect of HARP on <it>in vitro </it>and <it>in vivo </it>growth of PC-3 cells. This inhibition could be linked to a direct or indirect binding of this peptide to the HARP receptors (ALK, RPTPβ/ζ, nucleolin). <it>In vivo</it>, the P111-136 treatment significantly inhibits both the PC-3 tumour growth and the associated angiogenesis. Thus, P111-136 may be considered as an interesting pharmacological tool to interfere with tumour growth that has now to be evaluated in other cancer types.</p

A novel biweekly multidrug regimen of gemcitabine, oxaliplatin, 5-fluorouracil (5-FU), and folinic acid (FA) in pretreated patients with advanced colorectal carcinoma

Previous results suggest that GEM affects 5-fluorouracil (5-FU) metabolism and pharmacokinetics in cancer patients, while combined with oxaliplatin, levo-folinic acid, and 5-FU (GOLF regimen), at doses achievable in cancer patients, determines high cytotoxic and proapoptotic antitumour activity in colon cancer cells in vitro. On these bases we designed a phase I–II clinical trial testing the GOLF regimen in patients with metastatic colorectal carcinoma, who had received at least a prior line of chemotherapy. In total, 29 patients (20 males and nine females) enrolled in the study received every 2 weeks, gemcitabine (patients #1–3 received 600 mgm2; patients # 4–6 received 850 mgm2; while patients # 7–29 received 1000 mgm2) on the day 1, levo-folinic acid (100 mgm2) on the days 1 and 2; 5-fluorouracil (400 mgm2) in bolus injection, followed by a 22-h continuous infusion (800 mgm2) on the days 1 and 2, and oxaliplatin (85 mgm2), 6 h after the 5-FU bolus on day 2. The most frequent side effect was grade I–II haematological toxicity. In total, 28 patients were evaluable for response: three achieved a complete response, nine a partial response, 10 had a stable disease, and six progressed. The average time to progression and overall survival of the patients was, respectively, 7.26 and 22 months. Our GOLF combination is well tolerated and seems promising for the treatment of advanced colorectal cancer

Polymorphism in Gag Gene Cleavage Sites of HIV-1 Non-B Subtype and Virological Outcome of a First-Line Lopinavir/Ritonavir Single Drug Regimen

Virological failure on a boosted-protease inhibitor (PI/r) first-line triple combination is usually not associated with the detection of resistance mutations in the protease gene. Thus, other resistance pathways are being investigated. First-line PI/r monotherapy is the best model to investigate in vivo if the presence of mutations in the cleavage sites (CS) of gag gene prior to any antiretroviral treatment might influence PI/r efficacy. 83 patients were assigned to initiate antiretroviral treatment with first-line lopinavir/r monotherapy in the randomised Monark trial. We compared baseline sequence of gag CS between patients harbouring B or non-B HIV-1 subtype, and between those who achieved viral suppression and those who experienced virological failure while on LPV/r monotherapy up to Week 96. Baseline sequence of gag CS was available for 82/83 isolates; 81/82 carried at least one substitution in gag CS compared to HXB2 sequence. At baseline, non-B subtype isolates were significantly more likely to harbour mutations in gag CS than B subtype isolates (p<0.0001). Twenty-three patients experienced virological failure while on lopinavir/r monotherapy. The presence of more than two substitutions in p2/NC site at baseline significantly predicted virological failure (p = 0.0479), non-B subtype isolates being more likely to harbour more than two substitutions in this specific site. In conclusion, gag cleavage site was highly polymorphic in antiretroviral-naive patients harbouring a non-B HIV-1 strain. We show that pre-therapy mutations in gag cleavage site sequence were significantly associated with the virological outcome of a first-line LPV/r single drug regimen in the Monark trial

Regulator of G-protein signaling 18 controls both platelet generation and function

RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs) and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-). Interesting phenotypic differences between RGS18-/- and wild-type (WT) mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/- mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/- platelets displayed a higher sensitivity to activation in vitro. RGS18 deficiency markedly increased thrombus formation in vivo. In addition, RGS18-/- mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation in vitro and in vivo revealed a defective megakaryopoiesis in RGS18-/- mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery in vivo under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18

TIMP-1 Induces an EMT-Like Phenotypic Conversion in MDCK Cells Independent of Its MMP-Inhibitory Domain

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) regulate epithelial-mesenchymal transition (EMT) critical for the development of epithelial organs as well as cancer cell invasion. TIMP-1 is frequently overexpressed in several types of human cancers and serves as a prognostic marker. The present study investigates the roles of TIMP-1 on the EMT process and formation of the lumen-like structure in a 3D Matrigel culture of MDCK cells. We show that TIMP-1 overexpression effectively prevents cell polarization and acinar-like structure formation. TIMP-1 induces expression of the developmental EMT transcription factors such as SLUG, TWIST, ZEB1 and ZEB2, leading to downregulation of epithelial marker and upregulation of mesenchymal markers. Importantly, TIMP-1′s ability to induce the EMT-like process is independent of its MMP-inhibitory domain. To our surprise, TIMP-1 induces migratory and invasive properties in MDCK cells. Here, we present a novel finding that TIMP-1 signaling upregulates MT1-MMP and MMP-2 expression, and potentiates MT1-MMP activation of pro-MMP-2, contributing to tumor cell invasion. In spite of the fact that TIMP-1, as opposed to TIMP-2, does not interact with and inhibit MT1-MMP, TIMP-1 may act as a key regulator of MT1-MMP/MMP-2 axis. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule and also as an endogenous inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression