Protein B61 as a new growth factor: expression of B61 and up-regulation of its receptor epithelial cell kinase during melanoma progression.

Epithelial cell kinase (ECK) is a receptor protein tyrosine kinase, the role of which in melanoma biology is unclear. Here we studied the role of ECK during melanoma progression. ECK mRNA was overexpressed in virtually all melanoma lines tested, and levels were significantly higher in cell lines from distant metastases than primary melanomas; melanocytes were negative. Gene amplification was not detected in melanomas. Levels of ECK protein corresponded well with mRNA levels. B61 or LERK-1, recently identified as an ECK ligand, stimulated the growth of ECK-expressing melanoma cell lines, its first identified biological activity. Melanoma chemotaxis and chemoinvasion were not affected by B61. Growth of normal melanocytes was not affected. mRNA for B61 was detected in both melanoma cell lines and normal melanocytes. B61 was also identified by Western blotting and ECK binding activity with the use of a BIAcore binding assay in melanoma cell-conditioned media. These results suggest that B61 is an autocrine growth factor for melanomas but not normal melanocytes.

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Turnover and fate of plasma free fatty acids in briefly-fasted lymphoma-bearing mice.

Body fat loss during tumor growth may be due to increased mobilization of adipose triglycerides. Earlier work from this laboratory suggested that (i) lymphoma-bearing AKR mice have a circulating lipid mobilizing factor (LMF) which caused body fat loss during cancer growth; that (ii) fatty acids (FA) mobilized in these tumor-bearing (TB) mice were not oxidized to CO2 as in starved mice that lose their body fat; and that (iii) instead, the mobilized FA were sequestered by the lymphoma. We tested these hypotheses by injecting [1-14C]palmitate-albumin into lymphoma-bearing and control mice. We measured turnover of plasma FFA for 24 hr and predicted the cumulative conversion of tracer into breath 14CO2 (at 85 min) in the TB mice. Plasma FFA were mobilized more slowly in briefly fasted tumor-bearing mice than in controls with the same plasma FFA pool sizes. The fractional catabolic rate (FCR) (min-1) of plasma FFA turnover in both groups decreased during the night when the mice ate: postabsorptive controls, 1.07 (+/- 5.6%); fed controls, 0.25 (+/- 13%); postabsorptive TB, 0.53 (+/- 4.6%); fed TB, 0.29 (+/- 7.3%). Virtually all of the plasma FFA irreversible disposal in TB mice was accounted for as breath 14CO2 (30 to 40% I.D.), not as tumor lipids (1.1 +/- 0.22% I.D.). Thus, FFA oxidation to CO2 is the major fate of plasma FFA turnover in TB mice, and sequestration of FFA (palmitate) by tumor cells is a quantitatively minor process. The putative circulating LMF did not cause increased FFA mobilization in these lymphoma-bearing mice in the post-absorptive state.

Yeast vacuoles fragment when microtubules are disrupted.

To identify whether microtubules are involved in the maintenance of vacuolar morphology, we treated Saccharomyces cerevisiae with nocodazole and methyl benzimidazole-2-yl-carbamate, drugs which inhibit the polymerization of microtubules. Treated cells arrest with a single large bud in the G2/prophase portion of the cell cycle. Labeling the vacuole with either quinacrine or FITC-dextran revealed vacuole fragmentation that was not found in untreated cells or in cells arrested in G2 by unrelated means. A drug-resistant mutant in beta tubulin does not show vacuolar fragmentation when treated with drug. We propose that microtubules are involved in the regulation of vacuole morphology.

Delayed recycling of plasma FFA in mice: revised model of turnover and oxidation.

We have reexamined the concept of the irreversible disposal rate (IDR) or fractional catabolic rate (FCR) in relation to the oxidation of a major metabolic fuel, plasma free fatty acids (FFA), in mice. We measured the disappearance of intravenously injected [1-14C]palmitate complexed to mouse serum albumin from the circulation of normal mice fed ad libitum and fasted approximately 4 h before tracer injection. We also measured the appearance of expired 14CO2 in the breath. Using multicompartmental analyses (SAAM) we found that, in contrast to earlier studies in this and other mammals where the estimated IDR has always been two to three times greater than the oxidative rate, the IDR (FCR) corresponded closely to the fractional rate of plasma FFA oxidation to CO2. This rate could be estimated accurately in a study of 10- to 15-min duration. In an extended study (5-h duration) we obtained kinetic evidence of a major transport pathway that involves delayed recycling of FFA through at least several unidentified relatively slowly turning compartments.