Possible autocrine/paracrine actions of insulin-like growth factors during embryonic development: Expression and action of IGFs in undifferentiated P19 cells

The insulin-like growth factors I and II (IGF I and II) and their cell surface receptors are expressed in the mammalian embryo and may function as autocrine or paracrine growth factors during early development. P19 embryonic carcinoma cells, derived from a 7.5 day mouse embryo, were used as a model for a functional study of the IGF system in post-implantation embryogenesis. Undifferentiated P19 cells synthesized IGF I and II, the type I and II IGF receptors, and IGF binding proteins (IGF BP2, IGF BP3, and IGF BP4). P19 cells showed an increase in thymidine incorporation of 150% of control with a 4 hour incubation of IGF I (10 ng/ml) or IGF II (100 ng/ml) and an increase in cell viability compared to control cells during 24 hours of serum starvation. In both experiments IGF I was more potent than IGF II. Endogenous concentrations of IGF I and II in conditioned media were low compared to the doses of exogenous IGFs required for biologic effect, but nonetheless contributed significantly to baseline DNA synthesis, as demonstrated by inhibition of IGF actions with specific antibodies. Cell surface associated IGF BPs bound more radiolabeled IGF than IGF receptors, as determined by binding studies and affinity cross-linking. IGF I and IGF II appeared to regulate production of IGF BP2, suggesting that the IGFs may regulate their own actions by altering the abundance of their binding proteins. © 1993Wiley-Liss, Inc.     

Bones and genes: resolution problems in three Vietnamese species of Crocidura (Mammalia,Soricomorpha, Soricidae) and the description of?an?additional new species

Recent investigations of Southeast Asian white toothed shrews belonging to the genus Crocidura have revealed discrepancies between the results of morphological and molecular studies. The following study concerns three species of Crocidura occurring in Vietnam, namely Crocidura attenuata, Crocidura tanakae and Crocidura wuchihensis, and an undescribed fourth species revealed by molecular analysis. For many years Crocidura attenuata has been known to occur in Vietnam but, until very recently, the morphologically similar and comparably sized Crocidura tanakae was believed to be restricted to Taiwan. Following several molecular studies over the last few years, this species is now believed to be considerably more widespread and recognised as occuring also in Vietnam. The results of one of these recent molecular studies also revealed the presence of an undescribed species of Crocidura, similar in size and morphology to Crocidura wuchihensis, which is herein described. Data are provided on geographical variation in Vietnam and the problems of defining morphologically similar yet molecularly disparate species are discussed.     

Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization

The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G₀/G₁ phase of the cell cycle, the block occurs primarily at the G₂/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC₅₀ <1 μM. The pharmacokinetic study in BALB/c mice indicated good plasma exposure after intravenous administration. It was found to be efficacious at 1/10th the maximum-tolerated dose (1000 mg/kg) against human tumours derived from HCT-116 (colon) and NCI-H460 (lung) cells in SCID (severe-combined immunodeficient) mice models. BPT is a relatively better anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G₀/G₁. Its ability to effectively halt tumour growth in human tumour-bearing mice would suggest that BPT has the potential to be a candidate for further clinical development.A link between development of human cancers and cellular pathways where the retinoblastoma protein (pRb) has a major role is well established.^{1, 2} One of the frequent events associated with human tumour progression is abnormality in the pathway that links pRb, p16^INK4A, cyclin D1 and Cdk4 (cyclin-dependent kinase 4).³ Cdk4 along with its activating cyclin partner D1 has a key role in cell cycle control.^{4, 5} The naturally occurring inhibitor of Cdk4-cyclin D1, p16^INK4a (p16), is a tumour supressor protein. Deletion or inactivating mutations in the p16 gene are observed in many human cancers.^{6, 7} The catalytic activity of Cdk4 depends on its activation by the protein cyclin D1, which is expressed during the G₀/G₁ phase of the cell cycle. Many cancers are characterised by abnormal overproduction of cyclin D1.^{8, 9} As Cdk4 inhibitors target a pathway that links pRb, p16INK4A, cyclin D1 and Cdk4, it makes inhibition of Cdk4-cyclin D1 enzyme a crucially important target for cancer chemotherapy.^{10, 11, 12, 13} However, Rb mutations, consistent with loss of Rb function, have been identified in a wide spectrum of tumours including osteosarcomas, small-cell lung carcinomas, breast carcinomas and others, and the Cdk4 inhibitors cannot inhibit such pathway involving Rb-mutated tumours.A number of potential anticancer agents that selectively modulate the activity of Cdk4-cyclin D1 in vitro have been reported.¹⁴ These molecules also show the genotypic consequences of Cdk4 enzyme inhibition at the cellular level, that is, growth inhibition of cancer cells in vitro, arrest of asynchronous cells at G₀/G₁ and prevention of pRb phosphorylation at Cdk4-specific serine residues.^{15, 16, 17} Usually, competing with ATP molecules for binding at the protein kinase active site is the normal mechanism by which most small molecules inhibit kinase enzyme activity. Successful attempts to identify selective Cdk4 inhibitors using structure-based chemical design and molecular modelling have been reported.^{18, 19, 20} Furthermore, the success of Cdk4 inhibitors at clinical stages^{21, 22, 23, 24, 25} has indicated it as a promising therapeutic target for anticancer drug discovery.¹⁴Fascaplysin (1), a natural product originally isolated from a marine sponge, specifically inhibits the Cdk4 enzyme.^{26, 27} It inhibits Cdk4-cyclin D1 with an IC₅₀ of ~0.35 μM and blocks growth of cancer cells at the G₀/G₁ phase of the cell cycle. Similar to cryptolepine and ellipticine,²⁸ fascaplysin is also a planar structure and thus it intercalates double-stranded (d-s) DNA and shows unusual toxicity at the cellular level. It has been suggested that fascaplysin''s planar structure is the possible explanation for its ability to intercalate d-s DNA and also its unusual toxicity at the cellular level. To overcome this unusual toxicity, recently we reported CA224 (2), a non-planar analogue of fascaplysin exhibiting selective Cdk4 inhibition with no DNA-intercalating property.²⁹ In continuation to these efforts, herein we report identification of tryptoline-based compounds CA198 (3), CA199 (4), CA211 (5) and N-(biphenyl-2-yl)-tryptoline (BPT, 6) as selective Cdk4 inhibitors with no DNA-intercalating property. Based on the molecular modelling design, a number of non-planar analogues of fascaplysin were synthesized. They show specificity towards Cdk4-cyclin D1 enzyme activity and blocks the growth of cancer cells at the G₀/G₁ phase. Although BPT was also designed using a homology model of Cdk4, based on the X-ray crystallographic structures of Cdk2, Cdk4 and Cdk6,^{30, 31, 32, 33} further investigations showed that BPT blocks growth of cells at the G₂/M phase, in a Cdk-independent manner, through inhibition of tubulin polymerization. BPT shows potent cytotoxicity in a panel of cancer cells and is efficacious against human tumours derived from HCT-116 and NCI-H460 cells in SCID mice models. Here, we present the biological activity of BPT in detail. BPT (6) was synthesized using a one-step procedure by coupling tryptoline with biphenyl 2-carboxylic acid. The chemical synthesis of BPT and chemical structures of 1-6 are shown in Figure 1.Open in a separate windowFigure 1(a) Chemical structures of fascaplysin (1), CA224 (2) and its tryptoline analogues 3–6. (b) Synthetic scheme for BPT (6). (c) Molecular modelling studies to understand Cdk4 selectivity versus Cdk2: interactions of cis/trans conformations of BPT with Cdk2 and Cdk4, respectively (orange conformation is with Cdk2 and green with Cdk4)     

CXCR3 Signaling in BRAFWT Melanoma Increases IL-8 Expression and Tumorigenicity

Patients with early stage, radial growth phase (RGP) melanoma have a 97% survival rate; however, when the melanoma progresses to the invasive vertical growth phase (VGP), survival rates decrease to 15%. The targets of many clinical trials are the known genetic and molecular mechanisms involved in melanoma progression, with the most common oncogenic mutation being the BRAF^V600E. However, less than half of melanomas harbor this mutation, and consequently, do not respond to the current BRAF targeted treatments. It is therefore critical to elucidate alternative mechanisms regulating melanoma progression. Increased expression of the chemokine receptor, CXCR3, on melanoma cells is correlated with increased metastasis and poor patient outcomes, suggesting a role for CXCR3 in the RGP to VGP transition. We found that endogenous CXCR3 can be induced in two RGP cell lines, BOWES (BRAF^WT) and WM35 (BRAF^V600E), with in vitro environmental stress and nutrient deprivation. Signaling via induced endogenous CXCR3 is linked with IL-8 expression in BOWES cells. Ectopic overexpression of CXCR3 in BOWES cells leads to increased ligand-mediated phERK, cellular migration, and IL-8 expression in vitro, and to increased tumorigenesis and lymph node metastasis in vivo. Our results demonstrate that, in BRAF^WT melanomas, CXCR3 signaling mediates significant increases in IL-8 expression, suggesting that CXCR3 expression and signaling may represent a transformative event that drives the progression of BRAF^WT melanomas. Implications: Expression of CXCR3 on BRAF^WT melanoma cells may be a mediator of melanoma progression.     

Differential Impact of Acute High-Intensity Exercise on Circulating Endothelial Microparticles and Insulin Resistance between Overweight/Obese Males and Females

BackgroundAn acute bout of exercise can improve endothelial function and insulin sensitivity when measured on the day following exercise. Our aim was to compare acute high-intensity continuous exercise (HICE) to high-intensity interval exercise (HIIE) on circulating endothelial microparticles (EMPs) and insulin sensitivity in overweight/obese men and women.MethodsInactive males (BMI = 30 ± 3, 25 ± 6 yr, n = 6) and females (BMI = 28 ± 2, 21 ± 3 yr, n = 7) participated in three experimental trials in a randomized counterbalanced crossover design: 1) No exercise control (Control); 2) HICE (20 min cycling @ just above ventilatory threshold); 3) HIIE (10 X 1-min @ ∼90% peak aerobic power). Exercise conditions were matched for external work and diet was controlled post-exercise. Fasting blood samples were obtained ∼18 hr after each condition. CD62E⁺ and CD31⁺/CD42b^- EMPs were assessed by flow cytometry and insulin resistance (IR) was estimated by homeostasis model assessment (HOMA-IR).ResultsThere was a significant sex X exercise interaction for CD62E+ EMPs, CD31+/CD42b- EMPs, and HOMA-IR (all P<0.05). In males, both HICE and HIIE reduced EMPs compared to Control (P≤0.05). In females, HICE increased CD62E+ EMPs (P<0.05 vs. Control) whereas CD31+/CD42b- EMPs were unaltered by either exercise type. There was a significant increase in HOMA-IR in males but a decrease in females following HIIE compared to Control (P<0.05).ConclusionsOverweight/obese males and females appear to respond differently to acute bouts of high-intensity exercise. A single session of HICE and HIIE reduced circulating EMPs measured on the morning following exercise in males but in females CD62E+ EMPs were increased following HICE. Next day HOMA-IR paradoxically increased in males but was reduced in females following HIIE. Future research is needed to investigate mechanisms responsible for potential differential responses between males and females.     

Persistence and efficacy of a Beauveria bassiana biopesticide against the house fly,Musca domestica,on typical structural substrates of poultry houses

Entomopathogenic fungi, such as Beauveria bassiana, offer potential for use as biopesticides for control of house flies in poultry production facilities. This study evaluates persistence and efficacy of oil-formulated B. bassiana conidia against adult house flies on a range of structural substrates commonly found in poultry houses. Exposure of flies to fungal-treated surfaces produced high levels of infection leading up to 100% mortality in 6–10 days. However, the infectivity of the spray residues declined rapidly within 1 or 2 weeks following repeated fly exposures. Investigations showed that, in the absence of flies, conidia remained viable on test surfaces for up to 3 months regardless of substrate type, application method or fungal production batch. Rather, it was the presence of flies themselves that was responsible for reducing persistence. The exact mechanisms remain unclear but involve a combination of physical removal and chemical deactivation, with decay rates increasing at higher fly densities. While the rapid decay could pose a challenge for operational use, the results suggest it might be possible to tailor treatment frequencies to fly densities with, for example, weekly applications at high fly densities and longer intervals when populations decline. Further research is needed to determine persistence in semifield and field settings and to quantify the influence of fly densities under natural exposure conditions.