Intraoperative Imaging Techniques in Oncology.

Imaging-based procedures have become well integrated into the diagnosis and management of oncological patients and play a significant role in reducing morbidity and mortality rates. Here we describe the established and upcoming surgical oncological imaging techniques and their impact on cancer management.

The physical association of multiple molecular chaperone proteins with mutant p53 is altered by geldanamycin, an hsp90-binding agent.

Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin-proteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted that many transforming p53 mutants are found in stable physical association with molecular chaperones of the hsp70 class. To explore a possible role for aberrant chaperone interactions in mediating the altered function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a temperature-sensitive murine p53 mutant. In lysate prepared from A1-5 cells grown under mutant temperature conditions, hsp70 coprecipitated with p53Val135 as previously reported by others, but in addition, other well-recognized elements of the cellular chaperone machinery, including hsp90, cyclophilin 40, and p23, were detected. Under temperature conditions favoring wild-type p53 conformation, the coprecipitation of chaperone proteins with p53 was lost in conjunction with the restoration of its transcriptional activating activity. Chaperone interactions similar to those demonstrated in A1-5 cells under mutant conditions were also detected in human breast cancer cells expressing two different hot-spot mutations. To examine the effect of directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been shown to alter the chaperone associations regulating the function of unliganded steroid receptors. GA treatment of cells altered heteroprotein complex formation with several different mutant p53 species. It increased p53 turnover and resulted in nuclear translocation of the protein in A1-5 cells. GA did not, however, appear to restore wild-type transcriptional activating activity to mutant p53 proteins in either A1-5 cells or human breast cancer cell lines.

Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation.

Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.

Geldanamycin-stimulated destabilization of mutated p53 is mediated by the proteasome in vivo.

Mutation of the tumor suppressor gene p53 is the most common genetic abnormality detected in human cancers. Wild type p53 is a short-lived protein with very low basal intracellular levels. Most mutated forms of the protein, however, display markedly increased intracellular levels as an essential feature of their positive transforming activity. In this report, we have used selective inhibitors of the 20S proteasome to demonstrate that processing of p53 by ubiquitination and proteasome-mediated degradation is impaired by commonly occuring mutations of the protein. We found that this impairment of p53 turnover can be reversed by treatment of tumor cells with the benzoquinone ansamycin, geldanamycin, leading to a marked reduction in intracellular p53 levels. Finally, using cells which over-express a mutant p53 protein, we were able to demonstrate that restoration of proteasome-mediated degradation by geldanamycin is accompanied by p53 polyubiquitination. Although much remains to be learned about the mechanisms involved, our data demonstrate that selective de-stabilization of mutant transforming proteins such as p53 can be achieved pharmacologically with agents such as geldanamycin which modify the function of molecular chaperone proteins within tumor cells.

Mutant p53 protein expression interferes with p53-independent apoptotic pathways.

Loss of normal p53 function was found frequently to interfere with response of cancer cells to conventional anticancer therapies. Since more than half of all human cancers possess p53 mutations, we decided to explore the involvement of mutant p53 in drug induced apoptosis. To further evaluate the relationship between the p53-dependent and p53-independent apoptotic pathways, and to elucidate the function of mutant p53 in modulating these processes, we investigated the role of a p53 temperature-sensitive (ts) mutant in a number of apoptotic pathways induced by chemotherapeutic drugs that are currently used in cancer therapy. To that end, we studied the M1/2, myeloid p53 non-producer cells, and M1/2-derived temperature-sensitive mutant p53 expressing clones. Apoptosis caused by DNA damage induced with gamma-irradiation, doxorubicin or cisplatin, was enhanced in cells expressing wild type p53 as compared to that seen in parental p53 non-producer cells; mutant p53 expressing clones were found to be more resistant to apoptosis induced by these factors. Actinomycin D, a potent inhibitor of transcription, as well as a DNA damaging agent, abrogated the restraint apoptosis mediated by mutant p53. These observations suggest that while loss of wild type p53 function clearly reduces the rate of apoptosis, p53 mutations may result in a gain of function which significantly interferes with chemotherapy induced apoptosis. Therefore, to achieve a successful cancer therapy, it is critical to consider the specific relationship between a given mutation in p53 and the chemotherapy selected.

Basic microarray analysis: grouping and feature reduction.

DNA microarray technologies are useful for addressing a broad range of biological problems - including the measurement of mRNA expression levels in target cells. These studies typically produce large data sets that contain measurements on thousands of genes under hundreds of conditions. There is a critical need to summarize this data and to pick out the important details. The most common activities, therefore, are to group together microarray data and to reduce the number of features. Both of these activities can be done using only the raw microarray data (unsupervised methods) or using external information that provides labels for the microarray data (supervised methods). We briefly review supervised and unsupervised methods for grouping and reducing data in the context of a publicly available suite of tools called CLEAVER, and illustrate their application on a representative data set collected to study lymphoma.