A spatially and temporally restricted mouse model of soft tissue sarcoma.

Soft tissue sarcomas are mesenchymal tumors that are fatal in approximately one-third of patients. To explore mechanisms of sarcoma pathogenesis, we have generated a mouse model of soft tissue sarcoma. Intramuscular delivery of an adenovirus expressing Cre recombinase in mice with conditional mutations in Kras and Trp53 was sufficient to initiate high-grade sarcomas with myofibroblastic differentiation. Like human sarcomas, these tumors show a predilection for lung rather than lymph node metastasis. Using this model, we showed that a prototype handheld imaging device can identify residual tumor during intraoperative molecular imaging. Deletion of the Ink4a-Arf locus (Cdkn2a), but not Bak1 and Bax, could substitute for mutation of Trp53 in this model. Deletion of Bak1 and Bax, however, was able to substitute for mutation of Trp53 in the development of sinonasal adenocarcinoma. Therefore, the intrinsic pathway of apoptosis seems sufficient to mediate p53 tumor suppression in an epithelial cancer, but not in this model of soft tissue sarcoma.

Reversible amyloid formation by the p53 tetramerization domain and a cancer-associated mutant.

The tetramerization domain for wild-type p53 (p53tet-wt) and a p53 mutant, R337H (p53tet-R337H), associated with adrenocortical carcinoma (ACC) in children, can be converted from the soluble native state to amyloid-like fibrils under certain conditions. Circular dichroism, Fourier transform infrared spectroscopy and staining with Congo red and thioflavin T showed that p53tet-wt and p53tet-R337H adopt an alternative beta-sheet conformation (p53tet-wt-beta and p53tet-R337H-beta, respectively), characteristic of amyloid-like fibrils, when incubated at pH 4.0 and elevated temperatures. Electron micrographs showed that the alternative conformations for p53tet-wt (p53tet-wt-beta) and p53tet-R337H (p53tet-R337H-beta) were supramolecular structures best described as "molecular ribbons". FT-IR analysis demonstrated that the mechanism of amyloid-like fibril formation involved unfolding of the p53tet-wt beta-strands, followed by unfolding of the alpha-helices, followed finally by formation of beta-strand-containing structures that other methods showed were amyloid-like ribbons. The mutant, p53tet-R337H, had a significantly higher propensity to form amyloid-like fibrils. Both p53tet-wt (pH 4.0) and p53tet-R337H (pH 4.0 and 5.0), when incubated at room temperature (22 degrees C) for one month, were converted to molecular ribbons. In addition, p53tet-R337H, and not p53tet-wt, readily formed ribbons at pH 4.0 and 37 degrees C over 20 hours. Interestingly, unlike other amyloid-forming proteins, p53tet-wt-beta and p53tet-R337H-beta disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5. Although fibril formation at pH 4.0 was concentration and temperature-dependent, fibril disassembly at pH 8.5 was independent of both. Finally, we propose that the significantly higher propensity of the mutant to form ribbons, compared to the wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells.

The potential role of Fourier transform infrared spectroscopy and imaging in cancer diagnosis incorporating complex mathematical methods.

Infrared spectroscopy and imaging technology can provide new diagnostic capabilities for cancer research applications and to physicians directly involved in patient treatment. A methodology that permits the phenotypical description of cells and tissues is introduced, as well as a variety of new evaluation techniques that allow researchers and physicians to evaluate the infrared data at different levels of sophistication. The detection of cell alterations in the case of Chronic Lymphocytic Leukemia (CLL) demonstrates the capability of infrared spectroscopy to identify and stage this type of cancer, providing new prospects for diagnosis and treatment. The pathological study of oral tissue affected by Squamous Cell Carcinoma (SCC) illustrates the potential of Infrared Spectroscopy and Imaging for tissue diagnosis and cancer staging based in altered cell biochemistry, without using stains or any other marker technology. An example of combined fluid, cell and tissue analysis of thyroid cancers based on infrared technology is introduced to demonstrate the possibility of earlier detection of gland abnormalities and biochemical alterations in cell extracts using fine needle aspirates. Simple statistical techniques such as bivariate histogram analysis can distinguish between normal and altered cells and tissues when applied to infrared spectra and images. More complex mathematical techniques such as Principal Component Analysis (PCA) or Artificial Neural Networks (ANN) provide additional evaluation capabilities that can relate spectra of an unknown sample to an infrared reference database of known cell states. It is documented how this new infrared technology could enhance the diagnoses, treatment decisions and prognoses of patients in the field of cancer medicine.

Maturation of a tetravirus capsid alters the dynamic properties and creates a metastable complex.

The assembly of monomeric protein subunits into a viral capsid is a finely tuned molecular process. In response to subtle changes in environmental conditions, this supramolecular complex can dramatically reorganize. Defining the forces that control this structure and the cooperative action of subunits has implications for biology and nanotechnology. The small icosahedral RNA tetravirus family members Nudaurelia omega capensis (NomegaV) and Helicoverpa armigera stunt virus (HaSV) can be purified as provirions, and maturation to capsids can be induced by a drop in pH. In this study, a comparison of capsid secondary structure using FT-IR revealed that the procapsid has more alpha-helical content than the capsid, supporting the proposal that helix to coil transition may be important for maturation. The dynamic properties of the two states were probed using limited proteolysis and peptide mass mapping to identify regions of significant flexibility. Interestingly, the initial sites of protease cleavage were the N and C terminal domains that are internal in high-resolution models, and to inter-subunit surfaces. Further comparison of the two particle forms using FT-IR revealed that in response to thermal stress, the provirion disassembles and unfolds in a cooperative manner over a narrow temperature range (approximately 5 degrees C). Paradoxically, the capsid form, which is stable in a wide range of pH and ionic conditions and is more resistant to proteolysis, responds to thermal stress at a lower temperature than the procapsid form. This suggests that a metastable state is the end product of assembly.