Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR-ABL(+) leukemic cells to the abl inhibitor STI571.

BACKGROUND: Chronic myeloid leukemia is caused by a chromosomal translocation that results in an oncogenic fusion protein, Bcr-Abl. Bcr-Abl is a tyrosine kinase whose activity is inhibited by the antineoplastic drug STI571. This drug can cure mice given an injection of human leukemic cells, but treatment ultimately fails in animals that have large tumors when treatment is initiated. We created a mouse model to explore the mechanism of resistance in vivo. METHODS Nude mice were injected with KU812 Bcr-Abl(+) human leukemic cells. After 1 day (no evident tumors), 8 days, or 15 days (tumors >1 g), mice were treated with STI571 (160 mg/kg every 8 hours). Cells recovered from relapsing animals were used for in vitro experiments. Statistical tests were two-sided. RESULTS: Tumors regressed initially in all STI571-treated mice, but all mice treated 15 days after injection of tumor cells eventually relapsed. Relapsed animals did not respond to further STI571 treatment, and their Bcr-Abl kinase activity in vivo was not inhibited by STI571, despite high plasma concentrations of the drug. However, tumor cells from resistant animals were sensitive to STI571 in vitro, suggesting that a molecule in the plasma of relapsed animals may inactivate the drug. The plasma protein alpha1 acid glycoprotein (AGP) bound STI571 at physiologic concentrations in vitro and blocked the ability of STI571 to inhibit Bcr-Abl kinase activity in a dose-dependent manner. Plasma AGP concentrations were strongly associated with tumor load. Erythromycin competed with STI571 for AGP binding. When animals bearing large tumors were treated with STI571 alone or with a combination of STI571 and erythromycin, greater tumor reductions and better long-term tumor-free survival (10 of 12 versus one of 13 at day 180; P:<.001) were observed after the combination treatment. CONCLUSION: AGP in the plasma of relapsed animals binds to STI571, preventing this compound from inhibiting the Bcr/Abl tyrosine kinase. Molecules such as erythromycin that compete with STI571 for binding to AGP may enhance the therapeutic potential of this drug.

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification.

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic bcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/abl inhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 microM) 10-fold higher than the IC50 (0.1 microM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3-like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro. (Blood. 2000;95:1758-1766)

Activation of the adenovirus protease requires sequence elements from both ends of the activating peptide.

The adenovirus protease requires activation by an 11-residue peptide, GVQSLKRRRCF, to achieve maximum proteolytic activity. Derived from the C terminus of the viral protein pVI, the activating peptide (pVI-CT) forms a disulfide bond with cysteine 104 of the protease and causes a conformational change that accompanies the development of proteolytic activity. Results presented here show that the interaction of pVI-CT with the protease is dependent not only on the cysteine 10 but also on glycine 1 and valine 2. Removal of these residues, acetylation of the N-terminal glycine, or mutation of the valine to alanine or threonine significantly reduces or abolishes activation. Peptides lacking Gly-1 and Val-2 still form a disulfide bond with the protease but do not cause a conformational change in the protease also they are not effective inhibitors of activation as the interaction is readily reversed by full-length pVI-CT. These results suggest that pVI-CT causes activation by binding to two distinct regions of the protease and in doing so stabilizes the catalytic site. The reversible nature of the activation, suggested by the results presented here, may well reflect an in vivo regulatory mechanism.