Lonafarnib reduces the resistance of primitive quiescent CML cells to imatinib mesylate in vitro.

Recent studies indicate that a rare population of primitive quiescent BCR-ABL(+) cells are innately insensitive to imatinib mesylate (IM) and persist after IM therapy of patients with chronic myeloid leukemia (CML). New approaches to the eradication of these cells are therefore likely to be crucial to the development of curative therapies for CML. We have now found that Ara-C, LY294002 (a PI-3 (phosphatidylinositol-3' kinase) kinase inhibitor), 17AAG (a heat-shock protein (HSP)-90 antagonist) and lonafarnib (a farnesyltransfease inhibitor) all enhance the toxicity of IM on K562 cells and on the total CD34(+) leukemic cell population from chronic phase CML patients. However, for quiescent CD34(+) leukemic cells, this was achieved only by concomitant exposure of the cells to lonafarnib. Ara-C or LY294002 alone blocked the proliferation of these cells but did not kill them, and Ara-C, LY294002 or 17AAG in combination with IM enhanced the cytostatic effect of IM but did not prevent the subsequent regrowth of the surviving leukemic cells. These studies demonstrate the importance of in vitro testing of novel agents on the subset of primary leukemic cells most likely to determine long-term treatment outcomes in vivo.

Melanoma cell-derived factor stimulation of fibroblast glycosaminoglycan synthesis--the role of platelet-derived growth factor.

The hyaluronan-rich matrix surrounding many tumours may facilitate tumour growth, invasion and angiogenesis, with the majority of this hyaluronan apparently being synthesised by normal fibroblasts, stimulated to do so by tumour cell-derived factors. Melanoma cell-conditioned medium (CM) stimulates up to a 6-fold increase in fibroblast glycosaminoglycan (GAG) synthesis, with the active factors being present in tumour CM ultrafiltration fractions > 30 kDa and < 1 kDa. These fractions are poorly active individually, but when recombined, the activity is substantially greater than the additive effect. The objective of this study was to identify the factors present in the ultrafiltration fraction > 30 kDa that produce a greater than additive effect with the fraction < 1 kDa in stimulating the incorporation of 3H glucosamine into fibroblast GAGs. A number of factors including basic fibroblast growth factor (bFGF), interleukin (IL)-1 beta, pleiotrophin, platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha (TNF-alpha) and vascular endothelial growth factor (VEGF) failed to stimulate any significant increase in GAG synthesis, but when added to the < 1 kDa tumour CM fraction, both PDGF and to a lesser extent, bFGF, exhibited potent stimulating activities. Neutralising antibodies to PDGF and bFGF added to the melanoma CM decreased the fibroblast GAG-stimulating activity by 29% and 40%, respectively, in C8161 melanoma CM and by 47% and 45%, respectively, in Hs294T melanoma CM. The activities of PDGF-AA and PDGF-BB isoforms were indistinguishable, suggesting the PDGF-alpha receptor plays a role in the GAG-stimulatory response. Western analysis following treatment with PDGF, bFGF or melanoma CM revealed banding patterns for PDGF and melanoma CM that were similar. Immunoprecipitation of the PDGF-alpha receptor revealed it to be phosphorylated in fibroblasts treated with PDGF and melanoma CM, but not with control fibroblast CM. These studies suggest that PDGF plays an important role in the GAG-stimulating activity of the melanoma CM, but requires the presence of an as yet unidentified novel low molecular weight factor for full activity.

Effect of retinoic acid on melanoma cell-derived factor stimulation of fibroblast glycosaminoglycan synthesis.

The hyaluronan-rich matrix that surrounds many tumours and facilitates tumour cell growth and invasion is thought to be predominantly synthesized by normal stromal cells stimulated by tumour cell-derived factors. This study examines the possibility that the production of tumour cell-derived factors that stimulate fibroblast glycosaminoglycan (GAG) synthesis may be blocked by exposure to differentiation-inducing agents such as retinoic acid. We have demonstrated that Hs294T, C8161 and A375 human melanoma cell lines release factors into their medium that stimulate normal fibroblast GAG synthesis. Exposure of these melanoma cells to retinoic acid failed to mediate any significant reduction in growth over a 7-day period. Retinoic acid failed to block the tumour cell production of GAG-stimulating activities and even enhanced the activities produced by the C8161 cell line, particularly at low retinoic acid concentrations (48% stimulation at 10(-9) M retinoic acid; P < 0.02). Addition of retinoic acid directly to fibroblast cultures exposed to fibroblast-conditioned medium resulted in an inhibition of GAG synthesis with a 33% inhibition observed at 10(-5) M. Addition of retinoic acid to fibroblast cultures exposed to the tumour cell-conditioned medium failed to inhibit the stimulation of GAG synthesis. Other differentiation-inducing agents, such as hexamethylene-bis-acetamide and butyrate, also failed to block the production of tumour cell-derived GAG-stimulating activities. These results demonstrate that retinoic acid and other differentiation-inducing agents fail to inhibit melanoma cell production of fibroblast GAG synthesis-stimulating factors or their action upon fibroblasts.

Partial characterisation of human melanoma cell-derived factors that stimulate fibroblast glycosaminoglycan synthesis.

Glycosaminoglycans (GAGs), and in particular hyaluronan, are known to play a role in tumour cell migration, invasion and metastasis. Conditioned medium from two human metastatic melanoma cell lines (Hs294T and C8161) shows potent fibroblast GAG-synthesis-stimulating activities which are active in fibroblast cultures derived from different anatomical sites. This ability is not specific to melanoma cells and is observed in several carcinoma cell lines. Initial characterisation studies have demonstrated that the GAG-stimulating activities in the medium conditioned with melanoma cells show a degree of heat and trypsin resistance. Fractionation of the conditioned medium with Amicon ultrafiltration membranes of various molecular weight cut-offs, ranging from 1 to 30 kD, resulted in a total loss of activity. Activity could be regained by recombination of the concentrated fraction with the filtrate, suggesting more than one factor to be involved in GAG stimulation, with a degree of interdependence between the individual fractions. The fraction greater than 30 kD and that less than 1 kD appear to contain the majority of the GAG-stimulating activity.