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Biomedical subjects

Bruce Boman

Publications and source records attributed to Bruce Boman.

6 recordsLinked to original sources
PubMed2004

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection.

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.

Cell Line, Tumor↗
PubMed2004

Use of RNA amplification in the optimal characterization of global gene expression using cDNA microarrays.

Microarray analysis of human tissue is frequently hindered by the limited amount of RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total RNA derived from cultured renal epithelial cells from tuberous sclerosis complex (TSC) carriers and control individuals was amplified by in vitro transcription with T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified RNA (aRNA)) were analyzed as direct replicates and dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total RNA), 0.85 (40 microg total RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total RNA is proportional to the amount of RNA used and underscore the requirement of large amounts of total RNA for a comprehensive characterization of gene expression profiles. RNA amplification allows the detection of a large number of genes expressed in the starting RNA population without altering their relative intensities significantly. Thus, an RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of RNA, including those extracted from limiting clinical samples and microdissected histological specimens.

Gene Expression Profiling↗
PubMedDate not supplied

Analyzing alkaline proteins in human colon crypt proteome.

Normal human colon crypt protein extract was resolved by two-dimensional gel electrophoresis using pH 6-11 immobilized pH gradient strips in the first dimension. The optimized isoelectric focusing protocol includes cup-loading sample application at the anode and 1.2% hydroxyethyl disulfide (DeStreak), 15% 2-propanol and 5% glycerol in the rehydration buffer. Spots were well resolved across the entire pH range up to 11. A total of 311 protein spots were identified by mass spectrometry and peptide mass mapping. After combining isoforms, 231 nonredundant proteins were grouped into 16 categories according to their subcellular locations, and 17 categories according to their physiological functions. Histone proteins, ribosomal proteins and mitochondrial proteins were among the well-resolved highest p/ proteins. Application of this protocol to the analysis of normal and neoplastic colon crypts will contribute to the proteomic study of colorectal tumorigenesis since a significant portion of the human proteins is in basic pH range.

Cell Extracts↗
PubMedDate not supplied

Spontaneous immortalization of clinically normal colon-derived fibroblasts from a familial adenomatous polyposis patient.

Normal human diploid cells do not spontaneously immortalize in culture, but instead enter replicative senescence after a finite number of population doublings. Ablation of key checkpoint arrest or cancer-suppressor genes, through dominantly inherited germline mutation (p53+/-, Li-Fraumeni) or viral oncogene expression (SV40 large T, HPV16/18, and E6/E7) can lead to escape from senescence, additional doublings, and entrance into crisis phase, where immortal clones emerge at low frequency. In the vast majority of cases, telomerase is reactivated and telomeres are stabilized. Here we describe the spontaneous immortalization of clinically normal fibroblasts derived from colonic stroma of a familial adenomatous polyposis (FAP) patient. The preimmortal (C26C) and the spontaneously immortalized derivative (C26Ci) cells are heterozygous for a characterized germline mutation in exon 15 of the adenomatous polyposis coli gene. Immortalization was accompanied by spontaneous reactivation of endogenous telomerase and establishment of telomeres at presenescent lengths. Normal checkpoint behavior is retained and a diploid karyotype is maintained. These cells provide a valuable new addition to the limited number of spontaneously immortalized human cell types, particularly fibroblast cells, and will be useful in experimentally determining the functional pathways in neoplastic development and in the identification of potential molecular targets for cancer chemoprevention.

Adenomatous Polyposis Coli↗
PubMedDate not supplied

Microsatellite status and cell cycle associated markers in rectal cancer patients undergoing a combined regimen of 5-FU and CPT-11 chemotherapy and radiotherapy.

BACKGROUND: Microsatellite instability (MSI) and expression of cell cycle-related markers may predict a favorable outcome in colorectal cancer patients. The aim of this study was to elucidate the molecular profiles of patients with rectal cancers treated with neoadjuvant chemotherapy (5-Fluorouracil and CPT-11), radiotherapy and surgery that correlate with response to therapy. PATIENTS AND METHODS: Fifty-seven patients with rectal cancer were treated with the same preoperative chemotherapy regimen, radiotherapy (45 to 54 Gy) followed by surgery. The microsatellite status, the expression of the mismatch repair proteins MLH1 and MSH2 and p21WAF1/C1PI, p27, bcl-2, topoisomerase II (topo II) and Ki-67 were assessed in the pretreatment biopsies. The response to adjuvant therapy was categorized in the resected specimens as complete response (CR, no microscopic residual tumor present) and partial response (PR, tumor present). RESULTS: p21WAF1/C1PI, expression characterized the CR with 12 out of 30 tumors (40%) positive for this marker. None of the patients whose tumors did not express p21WAFI/C1PI (10 patients) was a CR (p=0.011). Overall, the tumors with CR also showed higher expression of bcl-2, Ki-67, topo II and p27. However, p53 was more frequently expressed in the PR tumors. Tumors with high microsatellite instability showed CR (3/5, 60%) more often than PR, whereas tumors with stable microsatellites showed PR (26/36, 80%) more often than CR (p=0.099). CONCLUSION: We conclude that a molecular profile characterized by high microsatellite instability with loss of mismatch repair protein expression and p21WAF1/C1PI is predictive of an improved response to neoadjuvant treatment with 5-FU, CPT-11 and radiation therapy.

Adult↗