Faculty Bibliography

A 10-year-old white boy presented clinically with thalassemia major facies, pallor, jaundice, and hepatomegaly. Investigation revealed the patient has hemoglobin (Hb) Lufkin concurrent with beta(0) thalassemia. DNA sequencing of the beta globin gene confirmed a GGC to a GAC mutation at codon 29 (gly to asp) for Hb Lufkin on the patient and also revealed a beta(0) thalassemia mutation, IVS-1-1 (G to A), on both the patient and his mother. Both parents lack the Hb Lufkin mutation. Molecular studies, human leukocyte antigen, and red blood cells phenotypic studies indicate spontaneous mutation of Hb Lufkin in this patient.

We identified a novel erythropoietin (Epo)-induced protein (CIP29) in lysates of human UT-7/Epo leukemia cells using two-dimensional gel analysis and cloned its full-length cDNA. CIP29 contains 210 amino acids with a predicted MW of 24 kDa, and has a N-terminal SAP DNA-binding motif. CIP29 expression was higher in cancer and fetal tissues than in normal adult tissues. CIP29 mRNA expression is cytokine regulated in hematopoietic cells, being up-regulated by Epo in UT7/Epo cells, and by thrombopoietin (Tpo), FLT3 ligand (FL) and stem cell factor (SCF) in primary human CD34(+) cells. Up-regulation of CIP29 in UT7/Epo cells by Epo was associated with cell cycle progression but not with antiapoptosis. Epo withdrawal reduced CIP29 expression concomitant with cell cycle arrest. Overexpression of CIP29-GFP in HEK293 cells enhances cell cycle progression. CIP29 appears to be a new cytokine regulated protein involved in normal and cancer cell proliferation.

BACKGROUND:The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival. RESULTS:We investigated the developmental expression of survivin in Pseudoglandular (approximately E14), Canalicular (approximately E15) and Terminal Bud (approximately E17) Stage SMGs. We report a significant 26% increase in transcript levels between the Canalicular and Terminal Bud Stages. Immunohistochemical studies demonstrate nuclear-localized survivin protein in epithelial cells bounding forming lumina in Canalicular and Terminal Bud Stage SMGs. CONCLUSIONS:Survivin is known to be a pro-survival and anti-apoptotic factor. Given that survivin translocation into the nucleus is required for the induction of entry into the cell cycle and the inhibition of apoptosis, our demonstration of nuclear-localized survivin protein in presumptive ductal and proacinar lumen-bounding cells suggests that survivin may be a key mediator of embryonic SMG epithelial cell survival.

We have investigated the interaction of the SH2-containing protein tyrosine phosphatase-1 (SHP-1) and Jak2 in an erythropoietin (Epo)-dependent human leukemia cell line, UT-7/Epo, using reciprocal immunoprecipitation and immunoblotting. The Epo-induced kinetics and dose response on phosphorylated Jak2 in anti-SHP-1 precipitates of UT-7/Epo cell lysates were similar to those in direct anti-Jak2 precipitates, suggesting that Jak2 coprecipitated with SHP-1. Furthermore, immunoblotting with anti-Jak2 and anti-SHP-1 antibodies indicated that SHP-1 appeared to be constitutively associated with non-tyrosine-phosphorylated Jak2 in UT-7/Epo cells in the absence of Epo and without phosphorylation of the Epo receptor (EpoR). Competition studies with C-terminal SHP-1 and Jak2 peptides decreased the amounts of SHP-1 and Jak2 detected in immunoprecipitates supporting the specific coprecipitation of SHP-1 and Jak2. In the presence of a recombinant GST-fusion protein containing both the N-terminal and C-terminal SH2 domains of SHP-1, anti-GST precipitated the fusion protein but not cellular Jak2. These studies suggest that SHP-1 and Jak2 are constitutively associated in UT-7/EPO cells. The association is not dependent upon Epo and is not mediated via SHP-1 SH2 binding. Sequential double immunoprecipitation demonstrated that only a small portion of intracellular Jak2 and SHP-1 molecules are constitutively associated. This partial association pattern may allow a more flexible and diverse regulation of Jak2 and SHP-1 activities. Whether Jak2 and SHP-1 are directly associated with each other or are part of a larger complex needs further investigation.