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Breast cancer is still one of the most important tumors among women in industrialized countries. Improvement in both understanding the molecular events associated with the disease and the development of new additional treatments is still an important goal to be achieved. Choline kinase (ChoK) is increased in human mammary tumors with high incidence, and this activation is associated with clinical variable indicators of greater malignancy. Here, we have investigated the role of ChoK in the development of breast cancer and found that ChoK is both necessary and sufficient for growth factor-induced proliferation in primary human mammary epithelial cells and an absolute requirement for the specific mitogenic response to heregulin in breast tumor-derived cells. These results demonstrate that ChoK plays an essential role in both normal human mammary epithelial cell proliferation and breast tumor progression. Furthermore, inhibition of ChoK shows a strong in vivo antitumor activity against human breast cancer xenografts. Thus, ChoK constitutes a novel bona fide molecular target for the treatment of breast cancer patients.

A cDNA corresponding to 16 kDa of the maize cyclin D2 N-terminus was cloned and this polypeptide was overexpressed to produce homologous antibodies. This antibody recognized a 38 kDa protein in extracts from maize embryonic axes which corresponds to the predicted size for cyclin D2 protein. Expression of cyclin D2 was followed at the transcriptional and protein levels, and the effect of cytokinins and abscisic acid (ABA) was followed during maize germination. Cytokinins importantly stimulated cyclin D2 gene expression at late germination times and sucrose was necessary for stimulation, whereas the effect of ABA was not different from that in controls. However, cyclin D2 protein levels in control axes reached a peak at 6 h germination, declining thereafter, and neither cytokinins nor ABA modified this behavior. Two cyclic-dependent kinase A (Cdk-A)-type proteins and proliferating cell nuclear antigen (PCNA) were found co-immunoprecipitating with cyclin D2, and these immunoprecipitates were able to phosphorylate both histone H1 and the maize retinoblastoma-related protein (RBR). This protein kinase activity differed from the pattern of protein accumulation during germination, and the activity was not modified by either cytokinins or ABA. We discuss these findings in terms of the importance of the cell cycle for the germination process.

Erythropoietin (EPO) increases the number of circulating erythrocytes and thus muscle oxygenation. The availability of the recombinant protein (rEPO) has increased the risk of its illegal use in sports, its detection being a difficult challenge. Five different hematopoietic parameters were initially chosen as indirect markers of rEPO abuse: concentration of serum EPO, concentration of serum-soluble transferrin receptors (sTFr), hematocrit, percentage of reticulocytes, and percentage of macrocytes. New models considering only hemoglobin, serum EPO concentration, and percentage of reticulocytes are simpler and seem to be more sensitive when low doses of rEPO are used. A more direct method of urine analysis (isoelectrofocusing, double blotting, and chemiluminescent detection) based on the charge differences between rEPO and endogenous EPO, related to their carbohydrate composition, provides proof of rEPO use. Furthermore, this approach permits the detection of darbepoetin, a direct analogue of EPO also known as NESP ("new erythropoiesis stimulating protein"). Recently a protein conjugate, "synthetic erythropoiesis protein" (SEP), containing precision-length, monodisperse, negatively charged polymers instead of oligosaccharides has been synthesized. Finally, EPO-mimetics are molecules capable of acting as EPO in dimerizing the EPO receptor. Two kinds of EPO-mimetics have been described: peptides and nonpeptides. The enhancement of oxygen availability to muscles by rEPO, analogues, and mimetics constitutes one of the main challenges to doping control. Major steps have already been developed for detection ofrEPO and some analogues. In the near future, the transfection to an athlete's body of genes that code for erythropoietin might be an emerging doping issue, and sports authorities have incorporated "gene doping" among the prohibited practices.