CREB is a transcription factor that functions in glucose homeostasis, growth factor-dependent cell survival, and memory. In this study, we describe a role of CREB in human cancer. CREB overexpression is associated with increased risk of relapse and decreased event-free survival. CREB levels are elevated in blast cells from patients with acute myeloid leukemia. To understand the role of CREB in leukemogenesis, we studied the biological consequences of CREB overexpression in primary human leukemia cells, leukemia cell lines, and transgenic mice. Our results demonstrate that CREB promotes abnormal proliferation and survival of myeloid cells in vitro and in vivo through upregulation of specific target genes. Thus, we report that CREB is implicated in myeloid cell transformation.
Sums-of-exponentials models are widely used in biomedical research, chiefly as models of data, despite a sizable folklore criticizing their usefulness. Problems in multiexponential model fitting are addressed here, along with an exposition of how to quantify them and critically assess their quality with available statistical methods and computer programs. This class of models also is reconciled with two classes of models of systems: multicompartmental and noncompartmental models. Key issues include the importance of choosing a correct data error model, the necessity for computing model precision estimates, and the distinction between problems due to experiment design or overparameterization and purported difficulties with multiexponential models. Methods for obtaining statistical estimates of model precision, for checking goodness of fit of competing models, and for improving sampling designs are presented. Also the classic Lanczos problem is revisited, and some difficulties are resolved with a more efficient experiment design.
We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of ؊200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.Urinary tract infection (UTI) is the most common urological disease in the United States and the second most common bacterial infection of any organ system (12, 32). UTI is a major cause of patient morbidity and health care expenditure for all age groups, accounting for over 7 million office visits and more than 1 million hospital admissions per year (39). Catheterassociated UTI accounts for 40% of all nosocomial infections and more than 1 million cases per year (22,41,49). The total cost of UTI to the United States health care system in the year 2000 was approximately $3.5 billion (13,19,20). An important component of these health care costs involves the processing of urine specimens by clinical microbiology laboratories. Urine is the type of body fluid most frequently submitted to clinical microbiology laboratories for culture. A major drawback of microbiological culture systems is the time lag of approximately 2 days between specimen collection and pathogen identification. The primary cause of the delay between specimen collection and pathogen identification is the time to colony formation after the specimen is plated on solid culture media.
The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr 253 and Tyr 257 ) in the ATP binding loop (P-loop) of BCR-ABL when Thr 315 is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr 315 mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.chronic myelogenous leukemia ͉ kinase inhibitor resistance ͉ phosphoproteomics ͉ imatinib ͉ dasatinib P oint mutations in the kinase domain of BCR-ABL are primarily responsible for resistance to the ABL inhibitor imatinib (Gleevec) in chronic myelogenous leukemia (CML) patients. The majority of imatinib-resistant BCR-ABL point mutations (of Ͼ50 distinct examples now reported clinically) impair drug binding by restricting flexibility of the enzyme, precluding adoption of the inactive conformation required for imatinib binding (1-4). The second generation Abl inhibitor dasatinib is effective against imatinib-resistant CML because it binds the BCR-ABL kinase domain regardless of activation loop conformation (5-7). As a result, the number of BCR-ABL mutations capable of conferring resistance to dasatinib is small and is limited almost exclusively to direct contact sites (8, 9). One mutation, T315I, confers resistance to imatinib, dasatinib, and the imatinib-related compound nilotinib (AMN-107) (10, 11).Although discovered in the context of drug resistance, there is growing evidence that these mutations may confer other fitness advantages to BCR-ABL. First, the T315I and E255K mutants were each detected by our group in imatinib-naïve CML blast crisis patients by direct sequencing of total BCR-ABL cDNA and are therefore estimated to account for at least 20% of the CML tumor burden in these patients (1). In addition, these and other kinase domain mutations have been identified before treatment in CML patients using mutation-specific quantitative PCR (12-18). Furthermore, the analogous mutation to T315I in the...
The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.
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