Here, we report the identification and characterization of a novel tyrosine phosphorylation site in the carboxy-terminal Src Homology 3 (SH3) (SH3C) domain of the Crk adaptor protein. Y251 is located in the highly conserved RT loop structure of the SH3C, a region of Crk involved in the allosteric regulation of the Abl kinase. Exploiting kinase assays to show that Y251 is phosphorylated by Abl in vitro, we generated affinity-purified antisera against phosphorylated Y251 in Crk and showed that Abl induces phosphorylation at Y251 in vivo, and that the kinetics of phosphorylation at Y251 and the negative regulatory Y221 site in vitro are similar. Y251 on endogenous Crk was robustly phosphorylated in chronic myelogenous leukemia cell lines and in A431 and MDA-MB-468 cells stimulated with epidermal growth factor. Using streptavidin–biotin pull downs and unbiased high-throughput Src Homology 2 (SH2) profiling approaches, we found that a pY251 phosphopeptide binds specifically to a subset of SH2 domains, including Abl and Arg SH2, and that binding of pY251 to Abl SH2 induces transactivation of Abl 1b. Finally, the Y251F Crk mutant significantly abrogates Abl transactivation in vitro and in vivo. These studies point to a yet unrealized positive regulatory role resulting from tyrosine phosphorylation of Crk, and identify a novel mechanism by which an adaptor protein activates a non-receptor tyrosine kinase by SH2 domain displacement.
Purpose. To compare the acute toxicities of IMRT to 3D-conformal radiation therapy (3DCRT) in the treatment of rectal cancer. Methods and Materials. Eighty-six patients with rectal cancer preoperatively treated with IMRT (n = 30) and 3DCRT (n = 56) were retrospectively reviewed. Rates of acute toxicity between IMRT and 3DCRT were compared for anorexia, dehydration, diarrhea, nausea, vomiting, weight loss, radiation dermatitis, fatigue, pain, urinary frequency, and blood counts. Fisher's exact test and chi-square analysis were applied to detect statistical differences in incidences of toxicity between these two groups of patients. Results. There were fewer hospitalizations and emergency department visits in the group treated with IMRT compared with 3DCRT (P = 0.005) and no treatment breaks with IMRT compared to 20% with 3DCRT (P = 0.0002). Patients treated with IMRT had a significant reduction in grade ≥3 toxicities versus grade ≤2 toxicities (P = 0.016) when compared to 3DCRT. The incidence of grade ≥3 diarrhea was 9% among 3DCRT patients compared to 3% among IMRT patients (P = 0.31). Conclusions. IMRT for rectal cancer can reduce treatment breaks, emergency department visits, hospitalizations, and all grade ≥3 toxicities compared to 3DCRT. Further evaluation and followup is warranted to determine late toxicities and long-term results of IMRT.
Despite the initial effectiveness of oncogene-directed cancer therapeutics, acquired drug resistance remains the ultimate “Achilles’ heel” for long-term durable remission in cancer patients. Acquisition of drug resistance is not more evident elsewhere than in the use of tyrosine kinase inhibitors (TKIs), Imatinib and Dasatinib, for patients with Chronic Myelogenous Leukemia (CML). Hence, while Imatinib initially produces remission in the chronic phase, ultimately these therapeutics fail via emergence of drug resistance, where CML can inevitably progress to a terminal blast phase culminating in a fatal outcome. Technically, it is challenging to predict the onset of drug resistance in a small number of oncogene-transformed cells, making the decision of when and how to employ second generation tyrosine kinase inhibitors, or employ novel compounds that would be of benefit in treating drug resistant Bcr-Abl mutants mainly retrospective. Here, we characterize a rapid and sensitive real-time Fluorescent Resonance Energy Transfer (FRET) based assay that is able to detect the in vivo activity of Bcr-Abl and its inhibition by small molecule compounds. Due to its real-time and in vivo nature, such an approach has the potential to monitor a drug-resistant phenotype, as well as to identify pharmaceutical agents that inhibit drug-resistant Bcr-Abl oncoproteins in vivo.
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