Efficacy of third-line therapy using bevacizumab in a patient with metastatic colorectal cancer.

Bevacizumab is currently approved in association with first- and second-line 5-fluorouracil-based chemotherapy regimens for patients with metastatic colorectal cancer. Few data about the usefulness of bevacizumab in third-line settings are available. We describe a patient refractory to FOLFIRI and FOLFOX chemotherapy regimens who showed a dramatic and durable response to bevacizumab and FOLFIRI. We also review and discuss the available literature.

Characterization of Cdk2-cyclin E complexes in plasma membrane and endosomes of liver parenchyma. Insulin-dependent regulation.

Rat liver parenchyma Golgi/endosomes fractions harbor a tyrosine-phosphorylated 34-kDa protein. Screening of Golgi, endosomes (ENs), plasmalemma (PM), and cytosolic (Cyt) fractions revealed the presence of the mitotic kinase Cdk2 in ENs, PM, and Cyt. The fluid phase endocytic marker horseradish peroxidase gained access to the endosomal Cdk2, confirming its localization. Cdk2 was shown to be associated to cyclin E and was active in ENs and PM fractions. The administration of a single dose of insulin (1.5 microgram/100 g, body weight) induced a time-dependent activation of the insulin receptor kinase in these structures. Insulin receptor-kinase activation was followed by the inhibition of immunoprecipitated Cdk2-cyclin E kinase activity in PM and the progressive disappearance of cyclin E. In marked contrast, no such effect was observed in ENs. The injection of a phosphotyrosyl phosphatase inhibitor (bpV(phen)) increased the levels of cyclin E in ENs and PM. A massive recruitment of p27(kip1) was observed in the Cdk2-cyclin E complexes isolated from PM and Cyt but not from ENs. In vitro, Cdk2-cyclin E complexes have the capacity to inhibit the formation of hybrid structures containing horseradish peroxidase and radioiodinated epidermal growth factor. Therefore, in the PM and ENs of adult rat liver, an active and regulated pool of the mitotic kinase Cdk2-cyclin E and some yet to be defined effectors are present. Cdk2 may contribute to the modulation of transport events and/or maintenance of the topology of endocytic elements.

Microtubule disruption induced in vivo by alkylation of beta-tubulin by 1-aryl-3-(2-chloroethyl)ureas, a novel class of soft alkylating agents.

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-isopropyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G2 transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3-2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC50 of 21.3+/-1.1 microM as compared with an IC50 of 11.6+/-0.7 microM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive beta-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-14C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional beta-tubulin band, indicating that CEU could covalently bind to the beta-tubulin. The 4-tBCEU-binding site on beta-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional beta-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site. To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the betaIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that betaIII-tubulin is not alkylated by [urea-14C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with beta-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of beta-tubulin.

Compartmentalization of the mitogen-activated protein kinase (MAPK) in hepatic endosomes: association with the internalized epidermal growth factor (EGF) receptor.

A pool of MAPK was found in hepatic plasma membrane (PM) and endosomes (ENs). After injection of a single dose of EGF (10 microg/100 g body weight), MAPK was detected in EGF receptor (EGFR) immunoprecipitates prepared from ENs. MAPK was detected in a time-dependent manner in EGFR immunoprecipitates that was coincident with the progressive concentration of the EGFR. The EGFR-associated MAPK was also detected by using an anti-phospho-MAPK suggesting that it was active. MAPK was present in wheat-germ agglutinin (WGA) eluates prepared from ENs and was maximally tyrosine-phosphorylated at the time peak of EGFR internalization. MAPK therefore is compartmentalized in PM and ENs of rat liver. A fraction of the endosomal MAPK was found to be associated with the internalized EGFR complexes, suggesting that it plays a role in the control of the EGFR activity at this locus.

Fibromuscular dysplasia of distal radial and ulnar arteries: uncommon cause of digital ischemia.

Fibromuscular dysplasia is an uncommon angiopathy that is principally observed in the renal and carotid arteries. Digital ischemia resulting from fibromuscular dysplasia of the forearm arteries is a rare occurrence. This article describes a case of distal radial and ulnar artery fibromuscular dysplasia presenting as paresthesia, claudication, and finger ulceration. Angiography was diagnostic in visualizing the characteristic "string of beads" appearance. In addition to the typical histological findings, we also observed a previously undescribed pathological finding. Surgical management involved resection of the diseased segment and primary anastomosis.

Effect of catecholamines on porcine Sertoli and Leydig cells in primary culture.

The accumulation by purified immature porcine Leydig and Sertoli cells of cyclic adenosine 3',5'-monophosphate in the presence of 1-methyl-3-isobuthylxathine was studied and their respective testosterone and 17 beta-estradiol production in response to catecholamines was assessed in vitro. These substances increased both basal and FSH-stimulated cyclic adenosine 3',5'-monophosphate accumulation in Sertoli cells. In contrast, catecholamines slightly enhanced basal cyclic adenosine 3',5'-monophosphate production but inhibited its human chorionic gonadotropin-stimulated accumulation by Leydig cells. Catecholamines had no effect on basal and stimulated testosterone release by these cells, while dopamine inhibited 17 beta-estradiol synthesis by Sertoli cells. Using various alpha- and beta-adrenergic agonists and antagonists, beta-receptors, likely of the beta 1-subtype, were shown to be present in both cell lines. Taken together these data suggest the presence of a cyclic adenosine 3',5'-monophosphate-linked adrenergic receptor in porcine Leydig and Sertoli cells, the role of which remains to be determined.

Isolation, purification and culture of Sertoli cells from immature piglet testes.

Several studies suggest a role of Sertoli cells in the control of Leydig cell steroidogenesis. In order to verify this hypothesis, we have developed a system for the purification of pig Sertoli cells. These cells were then characterized by their morphological appearance in light and electron microscopy, their ability to bind [125I]follicle stimulating hormone (FSH) and their functional capacity as evaluated by adenosine 3',5' monophosphate (cAMP) accumulation and lactate production when in primary culture under basal and FSH-stimulated conditions. Crude Sertoli cell suspensions from immature porcine testes were fractionated on discontinuous Percoll gradients (densities 1.025, 1.039, 1.055, 1.080 g/ml). Highly purified Sertoli cells were contained in the second band (d: 1.039) generated on the gradient. These cells demonstrated morphological and functional integrity as evidenced by binding specifically [125I] FSH and by responding to FSH stimulation (by an increased production of cAMP and lactate after 3 days in primary culture), but not to human chorionic gonadotrophin (hCG). This preparation represents a useful model for the study of Sertoli cell functions and their interation with Leydig cells in the regulation of testicular steroidogenesis.