A Reliable Method for the Selection of Exploitable Melanoma Archival Paraffin Embedded Tissues for Transcript Biomarker Profiling

The source tissue for biomarkers mRNA expression profiling of tumors has traditionally been fresh-frozen tissue. The adaptation of formalin-fixed, paraffin-embedded (FFPE) tissues for routine mRNA profiling would however be invaluable in view of their abundance and the clinical information related to them. However, their use in the clinic remains a challenge due to the poor quality of RNA extracted from such tissues. Here, we developed a method for the selection of melanoma archival paraffin-embedded tissues that can be reliably used for transcript biomarker profiling. For that, we used qRT-PCR to conduct a comparative study in matched pairs of frozen and FFPE melanoma tissues of the expression of 25 genes involved in angiogenesis/tumor invasion and 15 housekeeping genes. A classification method was developed that can select the samples with a good frozen/FFPE correlation and identify those that should be discarded on the basis of paraffin data for four reference genes only. We propose therefore a simple and inexpensive assay which improves reliability of mRNA profiling in FFPE samples by allowing the identification and analysis of “good” samples only. This assay which can be extended to other genes would however need validation at the clinical level and on independent tumor series

In Vitro Transformation of Primary Human CD34+ Cells by AML Fusion Oncogenes: Early Gene Expression Profiling Reveals Possible Drug Target in AML

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML

K+ efflux in deoxygenated sickle cells in the presence or absence of DIOA, a specific inhibitor of the [K+, Cl-] cotransport system

The ouabain bumetanide resistant (OBR) K+ efflux was investigated in deoxygenated sickle cells in comparison to oxygenated ones, by using a specific inhibitor of the [K+, Cl-] co-transport system, [(DihydroIndenyl)Oxy] Alkanoic acid (DIOA). A DIOA sensitive and a DIOA resistant K+ efflux were measured in deoxygenated sickle cells. The DIOA sensitive K+ efflux shared the properties of the [K+, Cl-] co-transport system, being stimulated by decreased pH and hypoosmolarity. This DIOA sensitive K+ efflux represented 70% of the total K+ efflux at pH 7.0 and at low pO2 (10-15 mmHg). Thus, a small reduction in Ph effectively stimulated the [K+, Cl-] co-transport system in deoxygenated condition, and this may contribute significantly to the sickle cell dehydration. We conclude that at pH lower than 7.4, the [K+, Cl-] co-transport system is permanently activated in sickle cells and leads to sickle cell dehydration in both oxygenated and deoxygenated conditions

Hemoglobin variants and activity of the (K+Cl-) cotransport system in human erythrocytes

To determine if the activation of the (K+Cl-) cotransport system observed in hemoglobin (Hb) S- or C-containing erythrocytes is related either to a global change of isoelectric point of the Hb molecule or to the specific location of these mutations on the position 6 of the beta chain of Hb, we studied the (K+Cl-) cotransport system in erythrocytes containing beta chain variants exhibiting either the Glu----Lys substitution observed in position beta 6 in Hb C (Hb E: beta 26 Glu----Lys; Hb O-Arab: beta 121 Glu----Lys; Hb Siriraj:beta 7 Glu----Lys) or the Glu----neutral residue substitution observed in position beta 6 in Hb S (Hb G-San Jose: beta 7 Glu----Gly; Hb D Punjab or D-Los Angeles: beta 121 Glu----Gln). The K transport mediated by the (K+Cl-) cotransport was increased in AC, AS and A-Siriraj and A-San Jose red blood cells and was similar to AA control in the other variants. These results indicate that an enhanced (K+Cl-) cotransport is not a property of all positively charged Hb variants, but it is mainly associated with mutations occurring at the beta 6 or beta 7 residues. An interaction of Hb with the cell membrane mediated by the disappearance of one of the negative charged residues (Glu) at this site of the A helix of the beta chain is the most likely candidate for the persistent activation of the (K+Cl-) cotransport system in these Hb variants