Inhibition of signal transduction by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin results in cytostasis and apoptosis.

17-Allylamino-17-demethoxygeldanamycin (17AAG) is a first-in-class heat shock protein 90 (Hsp90) molecular chaperone inhibitor to enter clinical trials. The downstream molecular and cellular consequences of Hsp90 inhibition are not well defined. 17AAG has shown activity against human colon cancer in cell culture and xenograft models. In this study, we demonstrated that in addition to depleting c-Raf-1 and inhibiting ERK-1/2 phosphorylation in human colon adenocarcinoma cells, 17AAG also depleted N-ras, Ki-ras, and c-Akt and inhibited phosphorylation of c-AKT: A consequence of these events was the induction of cell line-dependent cytostasis and apoptosis, although the latter did not result from dephosphorylation of proapoptotic BAD: One cell line, KM12, did not exhibit apoptosis and in contrast to the other cell lines overexpressed Bag-1, but did not express BAX: Taken together with other determinants of 17AAG sensitivity, these results should contribute to a more complete understanding of the molecular pharmacology of 17AAG, which in turn should aid the future rational clinical development and use of the drug in colon and other tumor types.

Inhibition of Kirsten-ras expression in human colorectal cancer using rationally selected Kirsten-ras antisense oligonucleotides.

Kirsten-ras is frequently mutated in colorectal cancers and may be an important therapeutic target, particularly because we have previously shown that acquisition of a mutation is associated with a poorer outcome. Understanding the role of Kirsten-ras and the consequences of inhibiting its activity or expression will contribute to our comprehension of colorectal cancer biology and may help to rationalize the choice of molecular targets suitable for therapeutic manipulation. Therefore we undertook a simple screen, incubating a library of oligonucleotides with Kirsten-ras mRNA and RNase H to identify an antisense oligonucleotide that effectively inhibited Kirsten-ras expression. We show for the first time in a human colon cancer cell line that inhibition of Kirsten-ras expression inhibits constitutive phosphorylation of Erk1/2, but not c-Akt, suggesting that in these cells constitutive phosphorylation of Erk 1/2 is dependent upon Kirsten-ras. Successful inhibition of Kirsten-ras had little effect on cell number or cell death and there was no evidence for accumulation of cells in any particular phase of the cell cycle. Kirsten-ras inhibition significantly reduced secretion of VEGF-A165 into the culture medium. Gene expression profiling by microarray detected altered expression of a number of genes. Of particular interest for future studies was the altered expression of genes encoding products involved in protein trafficking and the potential effects of these changes on cell adhesion. Our results suggest that, at least in this model, Kirsten-ras may contribute to malignancy predominantly through effects on angiogenesis, invasion, and metastasis, and that therapies directed at Kirsten-ras, including antisense approaches, may have particular utility through these mechanisms.

Magnetic resonance detects metabolic changes associated with chemotherapy-induced apoptosis.

Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects.

[Laboratory parameters as a guide for the radiologic study of infections of the urinary tract in childhood]. / I parametri di laboratorio come guida allo studio radiologico delle infezioni delle vie urinarie in età pediatrica.

Forty-nine children with urinary tract infection aged between 15 days and 10 years were studied. Thirty-three of them (67.3%) had urinary tract malformations. We valued the following parameters: a) White Blood Cells; b) Leukocyte differential; c) ESR at the first hour; d) CRP; e) Concentration ability; f) Body temperature. We wanted to investigate the usefulness of these parameters to predict which patients should be studied by X-ray of urinary tract. Single parameters did not prove very sensitive; the highest correlations were found with ESR and CRP, which were positive in 76% of the cases of malformations. Furthermore we valued the sensitivity and specificity of the tests grouped together: the highest values of sensitivity (97%) and specificity (31%) were obtained coupling the ESR and concentration ability.

[Vascular changes in postmastectomy "thick arm"]. / Studio delle modificazioni vascolari nel "braccio grosso" postmastectomia.

The authors, after shortly outlining some pathogenetic theories about postmastectomy "big arm", especially dwell upon the theory of the hindered venous discharge. They, subsequently, through a clear and interesting phlebographic iconography, illustrate ten of the 41 cases suffering from such syndrome, they recently had the opportunity to observe. On the basis of their own experience, and according to part of the literature thereabout, the authors think very probably the postmastectomy "big arm" is to be ascribed to the hindered venous reflux of upper limb (for thrombosis or compression), due to the operation of mastectomy. As a conclusion, they suggest the improvement or recovery of this syndrome by stepping over the stenosed venous tract through a by-pass.