The germline- and tissue-specific effects of endogenous point-mutant p53

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Vita.Includes bibliographical references.p53 is frequently altered in human tumors through missense mutations that result in accumulation of mutant p53 protein. These mutations may confer dominant-negative or gain-of- function properties to p53. To ascertain the physiological effects of tumor-associated point- mutations in p53, the structural mutant p53R172H and the contact mutant p53R270H (codons 175 and 273 in humans) were engineered into the endogenous p53 locus in mice. p53R270H/+ and p53R172H/+ mice are mouse models of Li-Fraumeni Syndrome (LFS). They developed allele- specific tumor spectra that were distinct from p53+/- mice and that better reflect the broad spectrum of tumors found in LFS patients. Dominant effects that varied by allele and function were observed in primary cells derived from these mice. In addition, p53R270H/- and p53R172H/- mice developed novel tumors compared to p53-/- mice, including hemangiosarcomas and variety of carcinomas. These data support a gain-of-function effect by mutant p53 toward the development of epithelial and endothelial tumors. Furthermore, conditional mutant p53 alleles were used in combination with a conditional activated K-ras allele to generate mouse models of advanced lung adenocarcinoma. In this system, the effects of endogenous mutant p53 were found to be both allele-specific and tissue- specific. This work provides insight into the spectrum of p53 mutations in human cancers and demonstrates that point-mutant p53 alleles expressed under physiological control have enhanced oncogenic potential beyond the simple loss ofp53 function.bu Kenneth Paul Olive.Ph.D

Generation and <i>ex vivo</i> culture of murine and human pancreatic ductal adenocarcinoma tissue slice explants

Traditional 2D/3D co-culture models typically do not reflect the cellular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) tumors, while in vivo models can be slow and ill-suited to mechanistic investigations. Here, we present a protocol for culturing murine PDAC explants and a corresponding human PDAC model using tissue slice explants. We describe steps for sponge production, preparation of media and materials, tissue collection, and sectioning. We then detail procedures for explant plating, daily culture, and collection of samples

Sharpening the predictions of big-bang nucleosynthesis

Motivated by the recent measurement of the primeval abundance of deuterium, we re-examine the nuclear inputs to big-bang nucleosynthesis (BBN). Using Monte-Carlo realization of the nuclear cross-section data to directly estimate the theoretical uncertainties for the yields of D, 3-He and 7-Li, we show that previous estimates were a factor of 2 too large. We sharpen the BBN determination of the baryon density based upon deuterium, rho_B = (3.6 +/- 0.4) * 10^{-31} g/cm^3 (Omega_B h^2 = 0.019 +/- 0.0024), which leads to a predicted 4-He abundance, Y_P = 0.246 +/- 0.0014 and a stringent limit to the equivalent number of light neutrino species: N_nu < 3.20 (all at 95% cl). The predicted 7-Li abundance, 7-Li/H = (3.5 + 1.1 - 0.9) * 10^{-10}, is higher than that observed in pop II stars, (1.7 +/- 0.3) * 10^{-10} (both, 95% cl). We identify key reactions and the energies where further work is needed.Comment: 5 pages, 4 figures (epsfig), REVTeX; submitted to Phys. Rev. Let

Pharmacological targeting of the receptor ALK inhibits tumorigenicity and overcomes chemoresistance in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients

Primordial nucleosynthesis with a varying fine structure constant: An improved estimate

We compute primordial light-element abundances for cases with fine structure constant alpha different from the present value, including many sources of alpha dependence neglected in previous calculations. Specifically, we consider contributions arising from Coulomb barrier penetration, photon coupling to nuclear currents, and the electromagnetic components of nuclear masses. We find the primordial abundances to depend more weakly on alpha than previously estimated, by up to a factor of 2 in the case of ^7Li. We discuss the constraints on variations in alpha from the individual abundance measurements and the uncertainties affecting these constraints. While the present best measurements of primordial D/H, ^4He/H, and ^7Li/H may be reconciled pairwise by adjusting alpha and the universal baryon density, no value of alpha allows all three to be accommodated simultaneously without consideration of systematic error. The combination of measured abundances with observations of acoustic peaks in the cosmic microwave background favors no change in alpha within the uncertainties.Comment: Phys. Rev. D accepted version; minor changes in response to refere

A novel method for quantification of gemcitabine and its metabolites 2',2'-difluorodeoxyuridine and gemcitabine triphosphate in tumour tissue by LC-MS/MS: comparison with (19)F NMR spectroscopy.

PURPOSE: To develop a sensitive analytical method to quantify gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and its metabolites 2',2'-difluorodeoxyuridine (dFdU) and 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) simultaneously from tumour tissue. METHODS: Pancreatic ductal adenocarcinoma tumour tissue from genetically engineered mouse models of pancreatic cancer (KP ( FL/FL ) C and KP ( R172H/+) C) was collected after dosing the mice with gemcitabine. (19)F NMR spectroscopy and LC-MS/MS protocols were optimised to detect gemcitabine and its metabolites in homogenates of the tumour tissue. RESULTS: A (19)F NMR protocol was developed, which was capable of distinguishing the three analytes in tumour homogenates. However, it required at least 100 mg of the tissue in question and a long acquisition time per sample, making it impractical for use in large PK/PD studies or clinical trials. The LC-MS/MS protocol was developed using porous graphitic carbon to separate the analytes, enabling simultaneous detection of all three analytes from as little as 10 mg of tissue, with a sensitivity for dFdCTP of 0.2 ng/mg tissue. Multiple pieces of tissue from single tumours were analysed, showing little intra-tumour variation in the concentrations of dFdC or dFdU (both intra- and extra-cellular). Intra-tumoural variation was observed in the concentration of dFdCTP, an intra-cellular metabolite, which may reflect regions of different cellularity within a tumour. CONCLUSION: We have developed a sensitive LC-MS/MS method capable of quantifying gemcitabine, dFdU and dFdCTP in pancreatic tumour tissue. The requirement for only 10 mg of tissue enables this protocol to be used to analyse multiple areas from a single tumour and to spare tissue for additional pharmacodynamic assays

Amyloid Precursor-like Protein 2 Expression Increases during Pancreatic Cancer Development and Shortens the Survival of a Spontaneous Mouse Model of Pancreatic Cancer.

In the United States, pancreatic cancer is a major cause of cancer-related deaths. Although substantial efforts have been made to understand pancreatic cancer biology and improve therapeutic efficacy, patients still face a bleak chance of survival. A greater understanding of pancreatic cancer development and the identification of novel treatment targets are desperately needed. Our analysis of gene expression data from patient samples showed an increase in amyloid precursor-like protein 2 (APLP2) expression within primary tumor epithelium relative to pancreatic intraepithelial neoplasia (PanIN) epithelial cells. Augmented expression of APLP2 in primary tumors compared to adjacent stroma was also observed. Genetically engineered mouse models of spontaneous pancreatic ductal adenocarcinoma were used to investigate APLP2\u27s role in cancer development. We found that APLP2 expression intensifies significantly during pancreatic cancer initiation and progression in the LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model, as shown by immunohistochemistry analysis. In studies utilizing pancreas-specific heterozygous and homozygous knockout of APLP2 in the KPC mouse model background, we observed significantly prolonged survival and reduced metastatic progression of pancreatic cancer. These results demonstrate the importance of APLP2 in pancreatic cancer initiation and metastasis and indicate that APLP2 should be considered a potential therapeutic target for this disease

Harmonic Motion Imaging for Abdominal Tumor Detection and High-Intensity Focused Ultrasound Ablation Monitoring: An In Vivo Feasibility Study in a Transgenic Mouse Model of Pancreatic Cancer

Abstract-Harmonic motion imaging (HMI) is a radiationforce-based elasticity imaging technique that tracks oscillatory tissue displacements induced by sinusoidal ultrasonic radiation force to assess the resulting oscillatory displacement denoting the underlying tissue stiffness. The objective of this study was to evaluate the feasibility of HMI in pancreatic tumor detection and high-intensity focused ultrasound (HIFU) treatment monitoring. The HMI system consisted of a focused ultrasound transducer, which generated sinusoidal radiation force to induce oscillatory tissue motion at 50 Hz, and a diagnostic ultrasound transducer, which detected the axial tissue displacements based on acquired radio-frequency signals using a 1-D cross-correlation algorithm. For pancreatic tumor detection, HMI images were generated for pancreatic tumors in transgenic mice and normal pancreases in wild-type mice. The obtained HMI images showed a high contrast between normal and malignant pancreases with an average peak-to-peak HMI displacement ratio of 3.2. Histological analysis showed that no tissue damage was associated with HMI when it was used for the sole purpose of elasticity imaging. For pancreatic tumor ablation monitoring, the focused ultrasound transducer was operated at a higher acoustic power and longer pulse length than that used in tumor detection to simultaneously induce HIFU thermal ablation and oscillatory tissue displacements, allowing HMI monitoring without interrupting tumor ablation. HMI monitoring of HIFU ablation found significant decreases in the peak-to-peak HMI displacements before and after HIFU ablation with a reduction rate ranging from 15.8% to 57.0%. The formation of thermal lesions after HIFU exposure was confirmed by histological analysis. This study demonstrated the feasibility of HMI in abdominal tumor detection and HIFU ablation monitoring

Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis

The existence of adult pancreatic progenitor cells has been debated. While some favor the concept of facultative progenitors involved in homeostasis and repair, neither a location nor markers for such cells have been defined. Using genetic lineage tracing, we show that Doublecortin-like kinase-1 (Dclk1) labels a rare population of long-lived, quiescent pancreatic cells. In vitro, Dclk1+ cells proliferate readily and sustain pancreatic organoid growth. In vivo, Dclk1+ cells are necessary for pancreatic regeneration following injury and chronic inflammation. Accordingly, their loss has detrimental effects after cerulein-induced pancreatitis. Expression of mutant Kras in Dclk1+ cells does not affect their quiescence or longevity. However, experimental pancreatitis converts Kras mutant Dclk1+ cells into potent cancer-initiating cells. As a potential effector of Kras, Dclk1 contributes functionally to the pathogenesis of pancreatic cancer. Taken together, these observations indicate that Dclk1 marks quiescent pancreatic progenitors that are candidates for the origin of pancreatic cancer