Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

Background: Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth. Methods:To quantitate NE properties of SCLCs, we developed a 50-gene expression-based NE score that could be applied to human SCLC tumors and cell lines, and genetically engineered mouse (GEM) models. We identified high and low NE subtypes of SCLC in all of our sample types, and characterized their properties. Results:We found that 16% of human SCLC tumors and 10% of SCLC cell lines were of the low NE

Cytoglobin (CYGB) is a recently discovered vertebrate globin distantly related to myoglobin with unknown function. CYGB is assigned to chromosomal region 17q25, which is frequently lost in multiple malignancies. Previous studies failed to detect evidence for mutations in the CYGB gene. Recent studies provided preliminary evidence for increased methylation of the gene in lung cancer. Our study was aimed at investigating the role of CYGB as a tumor suppressor gene. By nested methylation-specific DNA sequencing analysis of lung and breast cancer cell lines and bronchial and mammary epithelial cell lines, we identified that methylation of a 110-bp CpG-rich segment of the CYGB promoter was correlated with gene silencing. We specifically targeted this sequence and developed a quantitative methylation-specific PCR assay, suitable for high-throughput analysis. We showed that the tumor specificity of CYGB methylation in discriminating patients with and without lung cancer, using biopsies and sputum samples. We further showed the tumor specificity of this assay with multiple other epithelial and hematologic malignancies. To show tumor suppressor activity of CYGB, we performed the following: (a) RNA interference-mediated knockdown of CYGB gene on colony formation in a CYGB expressionpositive lung cancer cell line, resulting in increased colony formation; (b) enforced gene expression in CYGB expressionnegative lung and breast cancer cell lines, reducing colony formation; and (c) identification of potential proximate targets down-stream of the CYGB genes. Our data constitute the first direct functional evidence for CYGB, the newest member of the globin family, as a tumor suppressor gene.

The protooncogene c-kit is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. Homozygous mutations of this gene in the mouse cause anemia, infertility, and albinism, whereas heterozygous mutant mice usually exhibit only a white forehead blaze and depigmentation of the ventral body, tail, and feet. The heterozygous mouse phenotype is very similar to human piebald trait, which is characterized by a congenital white hair forelock and ventral and extremity depigmentation. To investigate the possibility that alterations in the human c-kit gene may be a cause of piebald trait, DNA from seven unrelated affected individuals was examined by Southern blot analysis. One subject, although cytogenetically normal, has a heterozygous deletion of the c-kit protooncogene. This deletion encompasses the entire coding region for c-kit and also involves the closely linked gene for platelet-derived growth factor receptor alpha. Fluorescence in situ hybridization of genomic c-kit probes to metaphase chromosomes independently confirmed the deletion in this case. These findings provide molecular evidence mapping piebald trait to the c-kit locus on chromosome 4. Although we cannot exclude the involvement of other closely linked genes, the demonstration of a genomic c-kit deletion in one subject with piebald trait and the marked concordance of the human and mouse phenotypes provide strong evidence for the role of c-kit in the development of human melanocytes and in the pathogenesis of piebald trait.

Detection of lung cancer at early stages could potentially increase survival rates. One promising approach is the application of suitable lung cancer-specific biomarkers to specimens obtained by non-invasive methods. Thus far, clinically useful biomarkers that have high sensitivity have proven elusive. Certain genes, which are involved in cellular pathways such as signal transduction, apoptosis, cell to cell communication, cell cycles and cytokine signaling are down-regulated in cancers and may be considered as potential tumor suppressor genes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. Using quantitative real time PCR, we tested 11 genes (3-OST-2, RASSF1A, DcR1, DcR2, P16, DAPK, APC, ECAD, HCAD, SOCS1, SOCS3) for levels of methylation within their promoter sequences in non-small cell lung cancers (NSCLC), adjacent non-malignant lung tissues, in peripheral blood mononuclear cells (PBMC) from cancer free patients, in sputum of cancer patients and controls. Of all the 11 genes tested 3-OST-2 showed the highest levels of promoter methylation in tumors combined with lowest levels of promoter methylation in control tissues. 3-OST-2 followed by, RASSF1A showed increased levels of methylation with advanced tumor stage (P<0.05). Thus, quantitative analysis of 3-OST-2 and RASSF1A methylation appears to be a promising biomarker assay for NSCLC and should be further explored in a clinical study. Our preliminary data on the analysis of sputum DNA specimens from cancer patients further support these observations.