Microphthalmia-associated transcription factor suppresses invasion by reducing intracellular GTP pools.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity.

Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.

Cellular delivery of hammerhead ribozymes conjugated to a transferrin receptor antibody.

Chemically-modified hammerhead ribozymes are sequence-specific RNA enzymes that can cleave target mRNA. These molecules have potential application as biological tools to understand gene expression and as therapeutic agents for the selective down-regulation of genes implicated in disease. However, as a result of their polyanionic character and relatively large molecular weights, ribozyme delivery to target cells is relatively inefficient. Using nuclease resistant 2'-O-methyl-modified ribozymes targeting the c-erbB1 oncogene, we have evaluated the potential use of human monoclonal transferrin-receptor antibody (TRA)-ribozyme conjugates for the improved delivery of ribozymes to A431 tumour cells. A 37-mer ribozyme derivatized with a free thiol-group at the 5'-end and bearing an internal [32P]-radiolabel was conjugated to either TRA or a non-specific IgG antibody using the heterobifunctional crosslinker, succinimidyl 4-(maleimido methyl)cyclohexane-1-carboxylate (SMCC). Up to six molecules of the ribozyme could be conjugated to one molecule of antibody. Cellular uptake studies in cultured human epidermoid A431 carcinoma cells showed that approximately a three-fold increase in cellular association could be obtained with the TRA-ribozyme conjugate compared to the free ribozyme. Cellular association of the conjugate was temperature-dependent and was inhibited by competition with excess free transferrin receptor antibody implying that conjugate uptake was consistent with the transferrin receptor-mediated endocytosis pathway. Treatment of cells with monensin further enhanced TRA-ribozyme conjugate cell association indicating that ribozyme delivery of conjugates may be further improved by strategies that modulate vesicular trafficking in cells.

An acute lower limb ischaemia with an unusual cause.

Acute arterial occlusion associated with an abdominal aortic aneurysm can occur as a result of acute thrombosis or embolism from a mural thrombus of the aneurysm. We report here a case of an unusual association between a seat belt injury and an acute ischaemic leg in a patient known to have an abdominal aortic aneurysm. We also demonstrate some difficult dilemma related to the use of anticoagulation in patients with multiple injuries.

The investigation and surgery of the post-thrombotic syndrome.

This paper reviews the problems encountered in the diagnosis and management of the post-thrombotic syndrome. The functional abnormalities found in this condition and the various methods available for their investigation are described. Past methods of treatment are reviewed and the necessity for objective functional assessment of new operations is stressed. The methods of operative treatment are outlined and the results so far reported are evaluated. The relative roles of surgery and conservative treatment are discussed.

Deep vein reflux: an assessment by descending phlebography.

This study of 51 patients aims to define the 'normal' range of reflux in the deep veins and the incidence of pathological reflux in patients with the post-thrombotic syndrome (PTS). 'Normal' limbs, limbs with simple varicose veins and limbs with post-thrombotic syndrome have been studied using a standardized technique for descending phlebography, supine and with a controlled Valsalva. The 'normal' range of reflux has been found to be grade 0-2. The incidence of pathological reflux in patients with proven post-thrombotic damage to the deep veins is 31 per cent.

Descending phlebography: a comparison of three methods and an assessment of the normal range of deep vein reflux.

Twenty-three legs in 13 patients without evidence of deep vein disease have been examined by descending phlebography using 3 techniques: with the patient semi-erect with no Valsalva manoeuvre with the patient semi-erect with a controlled Valsalva manoeuvre and with the patient supine with a standard Valsalva manoeuvre. It is concluded that the examination performed with the patient supine, using a standard Valsalva manoeuvre, is the most convenient and easiest to interpret of the variation. It is also suggested that deep vein reflux up to Grade 2 is within the normal range.

The etiology of the Klippel-Trenaunay syndrome.

The etiology of the Klippel-Trenaunay syndrome (KTS) remains obscure. Although venous hypertension secondary to deep venous obstruction has been suggested as a cause, recent studies have demonstrated that most patients have unimpeded venous drainage. Calf blood flows have been measured in 33 patients with KTS using venous occlusion plethysmography. Although all flow rates were within normal limits, flow in affected limbs was invariably greater than in normal limbs (p less than 0.001), and this is related to the presence of the nevus. Biopsies of subcutaneous veins demonstrate the histological features of a response to chronically raised flow. The authors suggest that KTS is caused by mesodermal abnormality during fetal development, leading to the maintenance of microscopic arteriovenous communications in the limb bud, as a result of which the triad of nevus, hypertrophy, and superficial varices is produced. Deep venous abnormalities occur pari passu with the triad and are not responsible for its development.

The Klippel-Trenaunay syndrome: clinical, radiological and haemodynamic features and management.

Forty-nine patients with the Klippel-Trenaunay syndrome have been studied. Sixty-eight per cent have a superficial, embryological venous channel on the lateral aspect of the limb, 25 per cent have had one or more severe spontaneous haemorrhages from dilated varices and 22 per cent have suffered a venous thrombo-embolism. Twenty-nine per cent have had episodes of rectal bleeding or haematuria associated with pelvic angiomas. Foot volumetry (n = 31) showed that calf emptying was decreased in only five patients and only one of these had phlebographic evidence of deep venous obstruction, which conflicts with previous findings. The rate of calf refilling was increased in 81 per cent and phlebography demonstrated incompetent communicating veins in 45 per cent of patients. No patient had clinical evidence of an arteriovenous fistula, and arteriography (n = 22) and calf blood flow (n = 33) were normal, but lymphangiography (n = 14) showed lymphatic hypoplasia in 55 per cent of limbs. Eighty-eight operations have been performed on 38 patients but symptoms persist in 90 per cent. We suggest that surgery should be limited to the excision of localized symptomatic abnormalities and that the best form of control is provided by graduated compression stockings.

A study of the mechanical properties of fresh and preserved human femoral vein wall and valve cusps.

In the hope that some varieties of the post-phlebitic syndrome might be treated by implanting a preserved vein valve, studies have been made of the mechanical properties of vein valves and vein wall before and after preservation with glutaraldehyde. The ultimate tensile strength (breaking stress) and strain (extensibility) of strips of vein wall and valve leaflet were measured with a Nene tensiometer. The ultimate tensile strength of valve leaflet was found to be twice that of vein wall. Preservation in glutaraldehyde (0.2 per cent, pH 7.4, for 7 days) with valve cusps closed by a minimum head of pressure caused no change in breaking stress or extensibility.

The diagnosis of aortic graft-enteric fistulae by computed tomography.

A number of techniques have been used to diagnose aortic graft-enteric fistulae. These are reviewed and their value assessed. Three patients are described in whom the diagnosis was correctly made from characteristic appearances seen on a computed tomography (CT) scan. Further experience may show that CT scanning has an important role in the detection of this condition.

The aetiology of cardiac arrest in patients undergoing surgical closure of an acute aorto-caval fistula.

Cardiac arrest during the repair of a leaking aortic aneurysm is a well recognized complication of an unsuspected aorto-caval fistula. Previous reports have suggested that death results from embolisation of debris through the fistula. We report a patient who underwent successful resuscitation in whom the sudden alteration in haemodynamics produced by crossclamping the aorta appeared responsible for the development of ventricular fibrillation. Preliminary caval compression with gradual application of the aortic clamp may allow time for equilibration to occur and prevent cardiac arrest.