Steven J Madore

The identification of E2F1-specific target genes

The E2F family of transcriptional regulators consists of six different members. Analysis of E2F-regulated promoters by using cultured cells suggests that E2Fs may have redundant functions. However, animal studies have shown that loss of individual E2Fs can have distinct biological consequences. Such seemingly conflicting results could be due to a difference in E2F-mediated regulation in cell culture vs. animals. Alternatively, there may be genes that are specifically regulated by an individual E2F which have not yet been identified. To investigate this possibility further, we have analyzed gene expression in E2F1 nullizygous mice. We found that loss of E2F1 did not cause changes in expression of known E2F target genes, suggesting that perhaps E2F1-specific promoters are distinct from known E2F target promoters. Therefore, we used oligonucleotide microarrays to identify mRNAs whose expression is altered on loss of E2F1. We demonstrate by chromatin immunoprecipitation that several of the promoters that drive expression of the deregulated mRNAs selectively recruit E2F1, but not other E2Fs, and this recruitment is via an element distinct from a consensus E2F binding site. To our knowledge, these are as yet undocumented examples of promoters being occupied in asynchronously growing cells by a single E2F family member. Interestingly, the E2F1-specific target genes that we identified encode proteins having functions quite different from the function of known E2F target genes. Thus, whereas E2F1 may share redundant functions in cell growth control with other E2F family members, it may also play an important biological role distinct from the other E2Fs.

Expression profiling and identification of novel genes in hepatocellular carcinomas.

Liver cancer is the fifth most common cancer worldwide and unlike certain other cancers, such as colon cancer, a mutational model has not yet been developed. We have performed gene expression profiling of normal and neoplastic livers in C3H/HeJ mice treated with diethylnitrosamine. Using oligonucleotide microarrays, we compared gene expression in liver tumors to three different states of the normal liver: quiescent adult, regenerating adult, and newborn. Although each comparison revealed hundreds of differentially expressed genes, only 22 genes were found to be deregulated in the tumors in all three comparisons. Three of these genes were examined in human hepatocellular carcinomas and were found to be upregulated. As a second method of analysis, we used Representational Difference Analysis (RDA) to clone mRNA fragments differentially expressed in liver tumors versus regenerating livers. We cloned several novel mRNAs that are differentially regulated in murine liver tumors. Here we report the sequence of a novel cDNA whose expression is upregulated in both murine and human hepatocellular carcinomas. Our results suggest that DEN-treated mice provide an excellent model for human hepatocellular carcinomas.

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation

In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox agents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability. Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants. Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA content. Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase. Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal. Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins.

Genetic evidence of a functional monocyte dichotomy.

Human peripheral blood monocytes are found as two distinct populations based upon differential expression of chemokine receptors, adhesion molecules, Fc receptors, and cytokines. cDNA microarray analysis now reveals additional differences between these subsets that suggest dramatically diverse functions. One monocyte subset (CD14++CD16−) appears to be closely paired with neutrophils, and may have as its primary function the removal and recycling of apoptotic neutrophils at sites of inflammation. The other monocyte subset (CD14+CD16+) expresses numerous genes encoding proteins with antimicrobial activity and thus may be more directly involved in peripheral host defense. The production of monocytes capable of efficiently removing dying neutrophils may be necessary to prevent host tissue damage and autoimmune response induction. Therefore, species like humans that produce relatively high levels of circulating neutrophils must also produce relatively high numbers of the recycling monocytes. Conversely, species such as mice and rats that maintain relatively lower levels of circulating neutrophils require fewer recycling monocytes.

The influence of tumor size and environment on gene expression in commonly used human tumor lines

BackgroundThe expression profiles of solid tumor models in rodents have been only minimally studied despite their extensive use to develop anticancer agents. We have applied RNA expression profiling using Affymetrix U95A GeneChips to address fundamental biological questions about human tumor lines.MethodsTo determine whether gene expression changed significantly as a tumor increased in size, we analyzed samples from two human colon carcinoma lines (Colo205 and HCT-116) at three different sizes (200 mg, 500 mg and 1000 mg). To investigate whether gene expression was influenced by the strain of mouse, tumor samples isolated from C.B-17 SCID and Nu/Nu mice were also compared. Finally, the gene expression differences between tissue culture and in vivo samples were investigated by comparing profiles from lines grown in both environments.ResultsMultidimensional scaling and analysis of variance demonstrated that the tumor lines were dramatically different from each other and that gene expression remained constant as the tumors increased in size. Statistical analysis revealed that 63 genes were differentially expressed due to the strain of mouse the tumor was grown in but the function of the encoded proteins did not link to any distinct biological pathways. Hierarchical clustering of tissue culture and xenograft samples demonstrated that for each individual tumor line, the in vivo and in vitro profiles were more similar to each other than any other profile. We identified 36 genes with a pattern of high expression in xenograft samples that encoded proteins involved in extracellular matrix, cell surface receptors and transcription factors. An additional 17 genes were identified with a pattern of high expression in tissue culture samples and encoded proteins involved in cell division, cell cycle and RNA production.ConclusionsThe environment a tumor line is grown in can have a significant effect on gene expression but tumor size has little or no effect for subcutaneously grown solid tumors. Furthermore, an individual tumor line has an RNA expression pattern that clearly defines it from other lines even when grown in different environments. This could be used as a quality control tool for preclinical oncology studies.