Bernard W Futscher

PMID: 24024150;PMCID: PMC3757676;Abstract:

Manganese superoxide dismutase, encoded by the Sod2 gene, is a ubiquitously expressed mitochondrial antioxidant enzyme that is essential for mammalian life. Mice born with constitutive genetic knockout of Sod2 do not survive the neonatal stage, which renders the longitudinal study of the biochemical and metabolic effects of Sod2 loss difficult. However, multiple studies have demonstrated that tissue-specific knockout of Sod2 in murine liver yields no observable gross pathology or injury to the mouse. We hypothesized that Sod2 loss may have sub-pathologic effects on liver biology, including the acquisition of reactive oxygen species-mediated mitochondrial DNA mutations. To evaluate this, we established and verified a hepatocyte-specific knockout of Sod2 in C57/B6 mice using Cre-LoxP recombination technology. We utilized deep sequencing to identify possible mutations in Sod2 mitochondrial DNA as compared to wt, and both RT-PCR and traditional biochemical assays to evaluate baseline differences in redox-sensitive pathways in Sod2^-/- hepatocytes. Surprisingly, no mutations in Sod2^-/- mitochondrial DNA were detected despite measurable increases in dihydroethidium staining in situ and concomitant decreases in complex II activity indicative of elevated superoxide in the Sod2^-/- hepatocytes. In contrast, numerous compensatory alterations in gene expression were identified that suggest hepatocytes havea remarkable capacity to adapt and overcome the loss of Sod2 through transcriptional means. Taken together, these results suggest that murine hepatocytes have a large reserve capacity to cope with the presence of additional mitochondrial reactive oxygen species. © 2013 The Authors.

PMID: 1352887;PMCID: PMC49508;Abstract:

An activated Ha-ras oncogene has been consistently found in chemically initiated benign and malignant mouse skin tumors, and an activated ras oncogene has been shown to initiate the process of mouse skin carcinogenesis. However, the exact timing of mutational activation of the Ha-ras gene relative to application of the chemical carcinogen is not known. A sensitive mutation-specific PCR technique was used to experimentally address the timing of Ha-ras gene mutational activation. This technique can detect mutant Ha-ras alleles in the presence of a very large excess of normal ras alleles. Activated Ha-ras genes with 61st codon A → T mutations were found in the epidermis of mice 1 week after topical initiation with 7,12-dimethylbenz[a]anthracene or urethane by using this assay. These results were confirmed by Xba I restriction fragment length polymorphism analysis and direct DNA sequencing. One week after initiation is 1-2 months before the appearance of benign papillomas that harbor activated Ha-ras oncogenes when the initiated mice are promoted with the tumor promoter phorbol 12-myristate 13-acetate. Our data support the hypothesis that initiated epidermal cells containing an activated Ha-ras gene can remain dormant in the skin until a tumor promoter induces regenerative hyperplasia that allows for outgrowth of these cells with an activated ras oncogene to give rise to a benign papilloma.

PMID: 11241756;Abstract:

Zearalenone is a naturally occurring estrogenic contaminant of moldy feeds and is present in high concentrations in dairy products and cereals. Zearalenone was postulated to contribute to the overall estrogen load of women, but the mechanisms of its action are not known. We demonstrated that zearalenone could stimulate the growth of estrogen receptor - positive human breast carcinoma cell line MCF-7. In addition, zearalenone functioned as an antiapoptotic agent by increasing the survival of MCF-7 cell cultures undergoing apoptosis caused by serum withdrawal. Treatment of these cells with 100 nM zearalenone induced cell-cycle transit after increases in the expression of c-myc mRNA and cyclins D1, A, and B1 and downregulation of p27^Kip-1. G1/G2-phase kinase activity and phosphorylation of the retinoblastoma gene product was also evident. Flow cytometric analysis demonstrated entry of cells into the S and G2/M phases of the cell cycle, and phosphorylation of histone H3 occurred 36 h after zearalenone treatment. Ectopic expression of a dominant-negative p21^ras completely abolished the zearalenone-induced DNA synthesis in these cells, and the specific inhibitor PD98059 for mitogen/extracellular-regulated protein kinase kinase arrested S-phase entry induced by zearalenone. These data suggest that the mitogen-activated protein kinase signaling cascade is required for zearalenone's effects on cell-cycle progression in MCF-7 cells. Given the presence of this mycotoxin in cereals, milk, and meat, the possibility that zearalenone is a potential promoter of breast cancer tumorigenesis should be investigated further. © 2001 Wiley-Liss, Inc.

PMID: 1511406;Abstract:

Cisplatin (cis-dichlorodiammineplatinum (II)) acts as a tumor initiator in the mouse skin model of carcinogenesis. DNA transfection studies suggested that skin tumors initiated by cisplatin contained dominant transforming activity. Since the Harvey-ras (H-ras) gene is known to be activated by point mutations in chemically initiated mouse skin tumors, we used polymerase chain reaction (PCR) and direct DNA sequencing to analyze the DNA sequence of the H-ras gene in twelve different cisplatin-initiated skin tumors. The results of these studies indicated that cisplatin-initiated skin tumors were normal (wild-type) at codons 12, 13, 61 and 117. Thus the transforming activity associated with cisplatin initiated mouse skin tumors was not due to a mutant H-ras gene and this suggests the involvement of other transforming genes during initiation of the mouse skin with cisplatin. © 1992.