Kristen A. Andersen

Cytotoxicity of Paclitaxel in Breast Cancer Is due to Chromosome Missegregation on Multipolar Spindles

The chemotherapy drug paclitaxel causes tumor regression and cell death by inducing high rates of chromosome missegregation on multipolar spindles. The Secret Life of Paclitaxel The classic chemotherapy drug paclitaxel is a standard part of treatment for breast cancer and other malignancies. Although it is commonly understood to act as a microtubule poison and lead to mitotic arrest, this knowledge is largely based on studies of cells in culture, with drug concentrations that may not be realistic. Now, Zasadil and coauthors measured the concentration of paclitaxel in real patients undergoing treatment with the drug, and then investigated the response of cancer cells to paclitaxel at these lower and more realistic concentrations. Unexpectedly, the cells treated under these conditions did not undergo mitotic arrest, but instead proceeded through mitosis with abnormal spindles, resulting in chromosome missegregation, which leads to tumor cell death. This intriguing discovery demonstrates that we may not know as much as we thought about the effects of one of our most common chemotherapy drugs. In addition, the findings from this study may lead to clinical applications both in optimizing the selection of chemotherapy drug combinations and in determining which patients are likely to respond to paclitaxel treatment. The blockbuster chemotherapy drug paclitaxel is widely presumed to cause cell death in tumors as a consequence of mitotic arrest, as it does at concentrations routinely used in cell culture. However, we determine here that paclitaxel levels in primary breast tumors are well below those required to elicit sustained mitotic arrest. Instead, cells in these lower concentrations of drug proceed through mitosis without substantial delay and divide their chromosomes on multipolar spindles, resulting in chromosome missegregation and cell death. Consistent with these cell culture data, most mitotic cells in primary human breast cancers contain multipolar spindles after paclitaxel treatment. Contrary to the previous hypothesis, we find that mitotic arrest is dispensable for tumor regression in patients. These results demonstrate that mitotic arrest is not responsible for the efficacy of paclitaxel, which occurs because of chromosome missegregation on highly abnormal, multipolar spindles. This mechanistic insight may be used to improve selection of future antimitotic drugs and to identify a biomarker with which to select patients likely to benefit from paclitaxel.

THE SIGMA-1 RECEPTOR IS ENRICHED IN POSTSYNAPTIC SITES OF C-TERMINALS IN MOUSE MOTONEURONS. AN ANATOMICAL AND BEHAVIORAL STUDY

The sigma-1 receptor regulates various ion channel activity and possesses protein chaperone function. Using an antibody against the full sequence of the sigma-1 receptor we detected immunostaining in wild type but not in knockout mice. The receptor was found primarily in motoneurons localized to the brainstem and spinal cord. At the subcellular level the receptor is restricted to large cholinergic postsynaptic densities on the soma of motoneurons and is colocalized with the Kv2.1 potassium channel and the muscarinic type 2 cholinergic receptor. Ultrastructural analysis of the neurons indicates that the immunostained receptor is located close but separate from the plasma membrane, possibly in subsurface cisternae formed from the endoplasmic reticulum (ER), which are a prominent feature of cholinergic postsynaptic densities. Behavioral testing on a rotorod revealed that Sigma-1 receptor knockout mice remained on the rotorod for significantly less time (a shorter latency period) compared to the wild type mice. Together these data indicate that the sigma-1 receptor may play a role in the regulation of motor behavior.

Diazo groups endure metabolism and enable chemoselectivity in cellulo.

We introduce a stabilized diazo group as a reporter for chemical biology. ManDiaz, which is a diazo derivative of N-acetylmannosamine, is found to endure cellular metabolism and label the surface of a mammalian cell. There its diazo group can undergo a 1,3-dipolar cycloaddition with a strained alkyne, providing a signal comparable to that from the azido congener, ManNAz. The chemoselectivity of diazo and alkynyl groups enables dual labeling of cells that is not possible with azido and alkynyl groups. Thus, the diazo group, which is approximately half the size of an azido group, provides unique opportunities for orthogonal labeling of cellular components.

Creating Site-Specific Isopeptide Linkages Between Proteins with the Traceless Staudinger Ligation

Site-specific isopeptide linkages between the ε-amino group of a lysine residue in one protein and a carboxyl group in another are central to ubiquitin-mediated protein degradation and other cellular processes. These linkages are inaccessible with common recombinant DNA techniques. Here, we describe a method to link two proteins by an authentic isopeptide bond. The method unites three techniques at the forefront of molecular biology. An azidonorleucine residue is installed at a desired site in a substrate protein by nonnatural amino acid incorporation, and a phosphinothioester is installed at the C terminus of a pendant protein by expressed protein ligation. Then, the traceless Staudinger ligation is used to link the substrate and pendant proteins via an isopeptide bond. This method facilitates the study of otherwise intractable protein structure-function relationships.

Intrinsic site-selectivity of ubiquitin dimer formation

The post‐translational modification of proteins with ubiquitin can take on many forms, including the decoration of substrates with polymeric ubiquitin chains. These chains are linked through one of the seven lysine residues in ubiquitin, with the potential to form a panoply of linkage combinations as the chain length increases. The ensuing structural diversity of modifications serves a variety of signaling functions. Still, some linkages are present at a much higher level than others in cellulo. Although ubiquitination is an enzyme‐catalyzed process, the large disparity of abundancies led us to the hypothesis that some linkages might be intrinsically faster to form than others, perhaps directing the course of enzyme evolution. Herein, we assess the kinetics of ubiquitin dimer formation in an enzyme‐free system by measuring the rate constants for thiol–disulfide interchange between appropriate ubiquitin variants. Remarkably, we find that the kinetically expedient linkages correlate with those that are most abundant in cellulo. As the abundant linkages also appear to function more broadly in cellulo, this correlation suggests that the more accessible chains were selected for global roles.