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Short hairpin RNA (shRNA)-expressing vectors have been shown stably to knock-down directed targets through RNA interference (RNAi). RNAi has emerged as a powerful tool for reverse genetic screens by assaying cellular phenotypes after exposure to libraries of pooled shRNAs directed against all or a subset of cellular mRNAs. Recently, noncoding RNAs have been recognized as major arbiters of cellular phenotypes. The scope and diversity of noncoding RNAs greatly enlarge the target pool for reversible genetic screens and underscore the desirability as screening tools of complex RNAi libraries, such as randomized RNAi libraries. We describe a novel approach to generate randomized shRNAs that takes advantage of a stable plasmid intermediate that arises in a FLP recombinase system. The ability of this system to generate randomized palindromes represents a new technology for shRNA generation including random shRNAs that cannot be produced synthetically. We describe this plasmid system and its use in generating randomized shRNAs from input 20-bp oligomers, and validate the functionality of a shRNA produced using this approach to knock-down estrogen receptor alpha expression.

Defining the molecular mechanisms that prevent myeloid progenitor cells from maturing is important because defects in maturation contribute to the development of myeloproliferative and myelodysplastic diseases. IL-3 is an important developmental factor for myeloid progenitor cells in vivo and is required to maintain the undifferentiated state in the 32Dcl3 cell line. The mechanisms employed by IL-3 to block differentiation, however, are not well understood. 32Dcl3 cells are myeloid progenitor cells of murine origin with high basal levels of p21waf1/cip1 (p21) expression. Our laboratory has previously reported that p21 levels decreased as CD34+-derived myeloid progenitor cells underwent terminal granulopoiesis in vitro. The effect of p21 upon the expression of genes associated with granulocytic differentiation has been unexplored, however. Since IL-3 maintains high levels of p21 in 32Dcl3 cells, we tested the hypothesis that p21 is an inhibitor of myeloid differentiation. Our findings demonstrate that siRNA knockdown of murine p21 is correlated with premature expression of the primary granule proteins myeloperoxidase and proteinase-3, proteins not abundant in cells maintained as myeloblasts by IL-3. Rescue with human p21 in these cells suppressed premature granule protein expression. p21 knockdown was also found to accelerate morphologic granulocytic differentiation in 32Dcl3 cells stimulated with G-CSF. Since high expression levels of p21 and overexpression of the IL-3 receptor have been correlated with poor outcomes in acute myeloid leukemias (AML), differentiation blockade by p21 may be one mechanism that contributes to AML pathogenesis.

The signal transducing protein Stat3 activates gene transcription in cells in response to multiple cytokines. Constitutive activation of Stat3 has been observed in solid tumors including head and neck squamous cell carcinoma. Stat3 activation in cancer has been associated with autocrine stimulatory loops and is believed to convey a growth advantage to cells. We now demonstrate ligand-independent activation of Stat3 by high cell density in multiple cancer cell lines. Activation of Stat3 is associated with antiproliferative rather than proliferative conditions. Interference with cdk2 activity upregulates Stat3 phosphorylation and Stat3-directed DNA-binding activity. Our data supports a model in which Stat3 activity is partially suppressed by cdk2 in growing cells and derepressed upon cell confluence.

Epidermal growth factor receptor (EGFR) is up-regulated and contributes to the loss of growth control in squamous cell carcinoma of the head and neck (SCCHN). Previously, we reported an association between autocrine stimulation of EGFR and constitutive signal transducers and activators of transcription (STAT) 3 activation in SCCHN cells in vitro and in vivo. Here, we evaluated the role of activated STAT3 in tumor progression and EGFR-independent mitogenic signaling. We found that SCCHN cells stably transfected with a dominant active STAT3 construct expressed elevated levels of STAT3 target genes, including Bcl-X(L) and cyclin D1, and demonstrated increased proliferation in vitro and more rapid tumor growth rates in vivo. Cell cycle analysis demonstrated an increased proportion of STAT3 construct transfectants in G(2)-M. These findings provide evidence that constitutive STAT3 activation contributes to tumor growth in SCCHN, independent of the EGFR autocrine axis.

The cell cycle behavior of hematopoietic cells varies from extended quiescence to spectacular proliferation. Cell cycle regulators choreograph these transitions through variation in the makeup of cyclin-dependent kinase (cdk)-containing complexes and through alteration in protein expression levels and subcellular localization. The mechanisms through which cell cycle regulators couple proliferation, differentiation and survival is coming into sharper focus. Cdk-inhibitors, once thought of solely in terms of a checkpoint function on cycling, are now known to interact directly with proteins and pathways central to differentiation and apoptosis. By shuttling between binding partners committed to discrete functional pathways, cell cycle regulators may directly coordinate proliferation with differentiation, migration and apoptosis.

Many of the therapeutic agents used for cancer chemotherapy today are based on decades-old agents which are highly cytotoxic but are nonselective for cancer cells. The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome translocation t(9;22), which results in the bcr-abl fusion gene. As an alteration that is nearly universal in CML cells, bcr-abl presents a therapeutic target that is unique to the pathological cells in CML patients. Advances in the understanding of the molecular mechanisms which sustain leukemic cells in CML have enabled the development of selective therapies for this disease. We review here the molecular pathogenesis and current treatment of CML. We also discuss the development of imatinib mesylate, a selective inhibitor of Bcr-Abl which has shown promise in clinical trials with CML. This recent advance in CML therapy represents a novel approach to rational design and development of new anticancer drugs.