Dean Billheimer

The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens.

PMID: 17549410;Abstract:

Knowledge of intrinsic tumor heterogeneity is vital for understanding of tumor progression mechanisms as well as for providing efficient treatments. In situ proteomic profiling of tumors is a powerful technology with potential to enhance our understanding of tumor biology, but sources of variability due to patient and tumor heterogeneity are poorly understood and are the topic of this investigation. Clarification of variability within case and between cases is also important for designing future studies. Direct protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a sensitive and powerful technology for obtaining hundreds of protein expression peaks from a thin tissue section. By combining robotic microspotting and laser capture microdissection with MALDI MS, we acquired multiple spectra per case to evaluate inter- and intra-case variability in human colorectal tumor and murine cecal carcinoma. We detected 256 peaks from 164 samples of 111 patients, which consisted of 55 normal colorectal mucosal samples, 24 adenomas, 71 primary carcinomas, and 14 hepatic metastases. In addition, we detected 291 peptide/protein peaks from 34 orthotopically transplanted murine cecal carcinomas and 14 hepatic metastases. In human colorectal samples, we observed that proteomic profiling in adenomas was more homogeneous across patients than in normal mucosa specimens (p=0.0008), but primary carcinoma exhibited greater heterogeneity than normal mucosa and adenomas (both p0.0001). Murine cecal carcinomas were homogeneous within and between carcinomas, while their hepatic metastases tended toward greater intra-tumor differences (p0.0001). Inter- and intra-case variability was approximately equal for many protein peaks. Acquiring up to 5 subsamples per case could reduce the total number of cases required, but further reduction from additional subsampling was modest unless intra-case variability comprises a greater proportion of total variation (e.g. >70%). In summary, this study characterizes intra- and inter-case variability of high-throughput protein expression in colorectal tumors, and provides guidance for the sample numbers required for in situ proteomic studies.

PMID: 21742769;Abstract:

Subcellular localization regulates BRCA1 function, and BRCA1 is exported to the cytoplasm following DNA damage in a p53-dependent manner. Because more than 50% of solid tumors harbor p53 mutations, it is possible that genetically wild-type (wt) BRCA1 is functionally abnormal through compromised nuclear-cytoplasmic shuttling in sporadic breast cancer patients with dysfunctional p53. In this study, we have investigated the mechanisms of p53-dependent BRCA1 subcellular distribution and DNA damage-induced nuclear export, as well as the impact on the resulting cytotoxic response to therapy in human breast cancer. We first show that p53 mediates BRCA1 nuclear export via protein - protein binding, rather than by modulation of its transcription. Furthermore, it is the C-terminal (BRCT) region of BRCA1 that is critical for its interaction with p53, and p53 may promote BRCA1 nuclear export by interrupting the association of BRCA1 with BARD1. In sporadic breast cancer specimens, dysfunctional p53 strongly correlates with nuclear retention of sequence-verified wt BRCA1. This p53-dependent BRCA1 shuttling determines cellular susceptibility to DNA damage as augmentation of cytosolic BRCA1 significantly enhances cancer cell susceptibility to ionizing radiation. Taken together, our data suggest that p53 dysfunction compromises nuclear export of wt BRCA1 as a mechanism to increase cellular resistance to DNA damage in sporadic breast cancer. We propose that targeting nuclear BRCA1 to the cytoplasm may offer a unique strategy to sensitize p53-deficient sporadic breast cancers to DNA damage - based therapy. ©2011 AACR.

PMID: 21357624;PMCID: PMC3108838;Abstract:

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633-641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20-30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.