Biomedical subjects

Charles W Caldwell

Publications and source records attributed to Charles W Caldwell.

5 recordsLinked to original sources

PubMed2003

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications.

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.

CpG Islands↗

PubMed2003

Methylation target array for rapid analysis of CpG island hypermethylation in multiple tissue genomes.

Hypermethylation of multiple CpG islands is a common event in cancer. To assess the prognostic values of this epigenetic alteration, we developed Methylation Target Array (MTA), derived from the concept of tissue microarray, for simultaneous analysis of DNA hypermethylation in hundreds of tissue genomes. In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 MTA amplicons, which represented the whole repertoire of methylated CpG islands in 93 breast tumors, 20 normal breast tissues, and 4 breast cancer cell lines, were arrayed on nylon membrane for probe hybridization. Positive hybridization signals detected in tumor amplicons, but not in normal amplicons, were indicative of aberrant hypermethylation in tumor samples. This is attributed to aberrant sites that were protected from methylation-sensitive restriction and were amplified by PCR in tumor samples, while the same sites were restricted and could not be amplified in normal samples. Hypermethylation frequencies of the 10 genes tested in breast tumors and cancer cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, 9% for DAPK1, and 0% for KL. Furthermore, hypermethylation of 5 to 7 loci of these genes was significantly correlated with hormone receptor status, clinical stages, and ages at diagnosis of the patients analyzed. This novel approach thus provides an additional avenue for assessing clinicopathological consequences of DNA hypermethylation in breast cancer.

Breast Neoplasms↗

PubMed2002

Expressed CpG island sequence tag microarray for dual screening of DNA hypermethylation and gene silencing in cancer cells.

We present a novel concept by using expressed CpG island sequence tags (ECISTs)for dual analysis of DNA methylation and gene expression in cancer cells. ECISTs are present in the genome and are DNA fragments expected to be located in the promoter and first exon region of genes. Their GC-rich segments can be used for screening hypermethylated CpG sites in cancer cells, and their exon-containing portions can be used for measuring levels of the corresponding transcripts. A total of 1162 loci met the criteria of ECISTs from an initial screening of 7776 CpG island tags. This ECIST panel was used to analyze the breast cancer cell line MDA-MB-231, which was treated with a demethylating agent. Microarray profile analysis identified 30 methylation-silenced genes, the expression of which could be directly reactivated by demethylation. An additional group of 96 up-regulated genes was also identified but appeared to be downstream from this epigenetic cascade. Thus, this study shows that the ECIST microarray can be used to differentiate the primary and secondary causes of demethylation and provides an effective tool to elucidate the mechanisms of aberrant DNA methylation in cancer.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed2002

Interpretation of flow cytometric measurement of lymphocytes after fluorescein angiography.

PURPOSE: By use of flow cytometry (FCM), lymphocyte subsets were evaluated with fluorochrome-labeled monoclonal antibodies (MoAbs). Recent fluorescein angiography (FA) produces temporary elevation of serum background fluorescence at certain wavelengths of light, producing falsely decreased lymphocyte subset quantitations. The authors evaluated the duration of this effect on one subset of lymphocytes after FA. METHODS: CD4 counts were determined by FCM before and 10 minutes, 1, 6, and 24 hours after injection of fluorescein dye in 12 patients. The MoAbs used were directly conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE). RESULTS: Using FITC-labeled MoAbs, falsely decreased CD4 counts occurred in all patients at 10 minutes and in all but one patient 1 hour after injection. Return to baseline levels occurred in 50% (95% confidence interval [CI], 0.21, 0.79) by 6 hours and in 75% (95% CI, 0.43, 0.95] by 24 hours. No such effect was observed using PE-labeled MoAbs. CONCLUSIONS: Falsely decreased CD4 values as determined by FCM were present immediately after FA in all 12 patients and persisted 24 hours in some patients when FITC-labeled MoAbs were used. CD4 evaluation should be delayed in patients who have undergone recent FA or the analysis should be performed with PE-labeled MoAbs.

Adult↗

PubMed2002

Cytohistologic discrepancies: a means to improve pathology practice and patient outcomes.

The use of cytohistologic discrepancies to investigate and reduce error seldom is studied. All gynecologic discrepancies (n = 283; 0.87% and 7.37% of all cytologic and histologic cases, respectively) and nongynecologic discrepancies (n = 146; 2.26% and 0.44% of all cytologic and histologic cases, respectively) for 26 months were classified as sampling or interpretive. Specimen type and pathologist discrepancy percentages, effect of discrepancies on patient outcome, and interobserver agreement of discrepancies were evaluated. Discrepancies were interpretive in 67% and 34% of gynecologic and nongynecologic cases, respectively. Statistically significant associations were seen between individual pathologist and discrepancy percentages. Breast (1.2%) and bronchial (0.8%) cytologic diagnoses had the highest discrepancy percentages. The kappa scores ranged from 0.02 to 0.45 for pairwise agreement of discrepant cases. Of nongynecologic interpretive discrepancies available for review, 63% (27/43) and 14% (6/43) were of no or minor clinical significance, respectively. Cytohistologic correlation is a useful tool to monitor performance and to identify specimen types prone to error.

Biopsy↗