A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics

The PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by the translocation, t(15;17) and the expression of a PML/RAR alpha fusion protein that is diagnostic of the disease. There is evidence that PML/RAR alpha protein acts as a dominant negative inhibitor of normal retinoid receptor function and myeloid differentiation. We now show that the PML/RAR alpha fusion product is directly downregulated in response to retinoic acid (tRA) treatment in the human APL cell line, NB4. tRA treatment induces loss of PML/RAR alpha at the protein level but not at the level of mRNA, as determined by Northern blots, by Western blots, and by ligand binding assays and in binding to RA-responsive DNA elements. We present evidence that this regulation is posttranslational. This evidence suggests that tRA induces synthesis of a protein that selectively degrades PML/RAR alpha. We further show that this loss of PML/ RAR-alpha is not limited to the unique APL cell line. NB4, because PML/RAR alpha protein is selectively downregulated by tRA when expressed in the transfected myeloid cell line U937. The loss of PML/RAR alpha may be directly linked to tRA-induced differentiation, because in a retinoid-resistant subclone of NB4, tRA does not decrease PML/RAR alpha protein expression. In NB4 cells, the specific downregulation of the fusion protein decreases the ratio of PML/RAR alpha to wild-type RAR alpha. Because the ratio of expression of PML/RAR alpha to wild-type RAR alpha and PML may be important in maintaining the dominant negative block of myelocytic differentiation, these data suggest a molecular mechanism for restoration by tRA normal myeloid differentiation in APL cells

The role of cell differentation in controlling cell multiplication and cancer

It has been suggested that cancer ought to be regarded as a disease of cell differentiation. In multicellular organisms, indeed, the control of cell multiplication is linked to cell specialization: During the process of differentiation embryonic cells, while cycling, acquire the ability to perform specialized functions. This ability is incompatible with cell cycling which, as a consequence, is repressed with forthcoming differentiation. Thus, the number of amplification divisions that occur during the period while differentiation is proceeding decides on the number of specialized cells produced. The progress in differentiation, contrary to usual assumptions, is accompanied by an increase in the cellular content of cytoplasm. The reason must be that cell specialization requires a specific amount and array of membrane-bounded cytoplasmic structures. Since the multiplication of these structures depends on membranous templates, their amount increases only if more cytoplasm is produced per cycle than required for a doubling, thus constituting an intracellular timer of differentiation: The larger the net rate of cytoplasmic growth per cell cycle, the fewer cycles occur. Extracellular signals modulate cell multiplication by altering the net rate of cytoplasmic growth. Each persisting alteration, however, that reduces this rate to zero, prevents differentiation, and thus causes the cells to retain embryonic capabilities and to initiate cancer. Cancer cells can be induced to differentiate and cease proliferation by support of cytoplasmic growth. This corroborates the suggestion that cancer must be regarded as a disease of cell differentiation and our conclusion that cancer is caused by a deficiency in cytoplasmic growth. Support of the latter must be an efficient principle in cancer therapy although limited by the organism's dependence on cell renewal