Considerations in the development of circulating tumor cell technology for clinical use.

This manuscript summarizes current thinking on the value and promise of evolving circulating tumor cell (CTC) technologies for cancer patient diagnosis, prognosis, and response to therapy, as well as accelerating oncologic drug development. Moving forward requires the application of the classic steps in biomarker development-analytical and clinical validation and clinical qualification for specific contexts of use. To that end, this review describes methods for interactive comparisons of proprietary new technologies, clinical trial designs, a clinical validation qualification strategy, and an approach for effectively carrying out this work through a public-private partnership that includes test developers, drug developers, clinical trialists, the US Food & Drug Administration (FDA) and the US National Cancer Institute (NCI).

Requirement for the SnoN oncoprotein in transforming growth factor beta-induced oncogenic transformation of fibroblast cells.

Transforming growth factor beta (TGF-beta) was originally identified by virtue of its ability to induce transformation of the AKR-2B and NRK fibroblasts but was later found to be a potent inhibitor of the growth of epithelial, endothelial, and lymphoid cells. Although the growth-inhibitory pathway of TGF-beta mediated by the Smad proteins is well studied, the signaling pathway leading to the transforming activity of TGF-beta in fibroblasts is not well understood. Here we show that SnoN, a member of the Ski family of oncoproteins, is required for TGF-beta-induced proliferation and transformation of AKR-2B and NRK fibroblasts. TGF-beta induces upregulation of snoN expression in both epithelial cells and fibroblasts through a common Smad-dependent mechanism. However, a strong and prolonged activation of snoN transcription that lasts for 8 to 24 h is detected only in these two fibroblast lines. This prolonged induction is mediated by Smad2 and appears to play an important role in the transformation of both AKR-2B and NRK cells. Reduction of snoN expression by small interfering RNA or shortening of the duration of snoN induction by a pharmacological inhibitor impaired TGF-beta-induced anchorage-independent growth of AKR-2B cells. Interestingly, Smad2 and Smad3 play opposite roles in regulating snoN expression in both fibroblasts and epithelial cells. The Smad2/Smad4 complex activates snoN transcription by direct binding to the TGF-beta-responsive element in the snoN promoter, while the Smad3/Smad4 complex inhibits it through a novel Smad inhibitory site. Mutations of Smad4 that render it defective in heterodimerization with Smad3, which are found in many human cancers, convert the activity of Smad3 on the snoN promoter from inhibitory to stimulatory, resulting in increased snoN expression in cancer cells. Thus, we demonstrate a novel role of SnoN in the transforming activity of TGF-beta in fibroblasts and also uncovered a mechanism for the elevated SnoN expression in some human cancer cells.

P-selectin glycoprotein ligand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates rolling of leukocytes on P-selectin under flow. The glycoproteins that enable leukocyte tethering to or rolling on E-selectin are not known. We used gene targeting to prepare PSGL-1-deficient (PSGL-1-/-) mice, which were healthy but had moderately elevated total blood leukocytes. Fluid-phase E-selectin bound to approximately 70% fewer sites on PSGL-1-/- than PSGL-1+/+ neutrophils. Compared with PSGL-1+/+ leukocytes, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in vitro, because their initial tethering to E-selectin was impaired. The residual cells that tethered rolled with the same shear resistance and velocities as PSGL-1+/+ leukocytes. Compared with PSGL-1+/+ mice, significantly fewer PSGL-1-/- leukocytes rolled on E-selectin in TNF-alpha-treated venules of cremaster muscle in which P-selectin function was blocked by an mAb. The residual PSGL-1-/- leukocytes that tethered rolled with slow velocities equivalent to those of PSGL-1+/+ leukocytes. These results reveal a novel function for PSGL-1 in tethering leukocytes to E-selectin under flow.

DMD(mdx3Cv) and DMD(mdx4Cv) dystrophin mutations in mice: rapid polymerase chain reaction genotyping.

We devised non-radioactive PCR assays for the DMD(mdx3Cv) and DMD(mdx4Cv) mouse dystrophin point mutations, in which mutant and wild type reactions electrophoresed separately diagnose whether the DNA carries the mutant, wild type, or both alleles. This simple and reliable assay facilitates the use of these mutant mouse models, which have an extended inflammatory phase (DMD(mdx3Cv)), less reversion to wild type (DMD(mdx4Cv)), and reduced expression of dystrophin mRNAs arising from internal promoter usage than the DMD(mdx) mouse. The PCR assays described facilitate the use of the DMD(mdx3Cv) and DMD(mdx4Cv) mutant mouse models, when maintaining the mutations as heterozygotes, backcrossing into different inbred genetic backgrounds, or when crossing targeted mutations into these dystrophic backgrounds.

Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720.

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.