Altering mammalian transcription networking with ADAADi: An inhibitor of ATP-dependent chromatin remodeling.

Active DNA-dependent ATPase A Domain inhibitor (ADAADi) is the only known inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. The molecule is synthesized by aminoglycoside phosphotransferase enzyme in the presence of aminoglycosides. ADAADi interacts with ATP-dependent chromatin remodeling proteins through motif Ia present in the conserved helicase domain, and thus, can potentially inhibit all members of this family of proteins. We show that mammalian cells are sensitive to ADAADi but with variable responses in different cell lines. ADAADi can be generated from a wide variety of aminoglycosides; however, cells showed differential response to ADAADi generated from various aminoglycosides. Using HeLa and DU145 cells as model system we have explored the effect of ADAADi on cellular functions. We show that the transcriptional network of a cell type is altered when treated with sub-lethal concentration of ADAADi. Although ADAADi has no known effects on DNA chemical and structural integrity, expression of DNA-damage response genes was altered. The transcripts encoding for the pro-apoptotic proteins were found to be upregulated while the anti-apoptotic genes were found to be downregulated. This was accompanied by increased apoptosis leading us to hypothesize that the ADAADi treatment promotes apoptotic-type of cell death by upregulating the transcription of pro-apoptotic genes. ADAADi also inhibited migration of cells as well as their colony forming ability leading us to conclude that the compound has effective anti-tumor properties.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation.

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer.

Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells.

Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer.

A preliminary study on the occurrence of keratinophilic fungi in soils of Jamaica.

This report represents the first study of keratinophilic fungi present in soils of Jamaica. Out of the 40 soil samples examined from different habitats, 30 (75%) were positive for the presence of keratinophilic fungi, yielding 36 isolates of keratinophilic fungi. Microsporum gypseum complex (represented by 16 isolates of M. gypseum, and four of M. fulvum) was most frequent, being present in 50% of the samples. A very high occurrence of this dermatophyte in Jamaican soil is of public health significance. The remaining isolates of keratinophilic fungi were represented by Chrysosporium spp (mainly C. indicum and C. tropicum) and Sepedonium sp.

A preliminary suudy on the occurrence of keratinophilic fungi in soils of Jamaica / Estudo preliminar sobre a ocorrência de fungos queratinofílicos em solos da Jamaica

This report represents the first study of keratinophilic fungi present in soils of Jamaica. Out of the 40 soil samples examined from different habitats, 30 (75%) were positive for the presence of keratinophilic fungi, yielding 36 isolates of keratinophilic fungi. Microsporum gypseum complex (represented by 16 isolates of M. gypseum, and four of M. fulvum) was most frequent, being present in 50% of the samples. A very high occurrence of this dermatophyte in Jamaican soil is of public health significance. The remaining isolates of keratinophilic fungi were represented by Chrysosporium spp (mainly C. indicum and C. tropicum) and Sepedonium sp.

Esta comunicação representa o primeiro estudo sobre fungos queratinofílicos presentes em solos da Jamaica. De 40 amostras de solo examinadas de diferentes localidades, 30 (75%) foram positivas para a presença de fungos queratinofílicos permitindo 36 isolamentos dos mesmos. O complexo Microsporum gypseum (representados por 16 isolamentos de M. gypseum e quatro de M. fulvum) foi o mais frequente, estando presente em 50% das amostras. A muito alta ocorrência deste dermatófito no solo da Jamaica é significante para a saúde pública. Os isolados remanescentes de fungos queratinofílicos foram representados pelo Chrysosporium spp (principalmente C. indicum e C. tropicum) e Sepedonium sp.

Global epigenetic changes induced by SWI2/SNF2 inhibitors characterize neomycin-resistant mammalian cells.

BACKGROUND: Previously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes. METHODOLOGY: Using fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase ADomain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized. SIGNIFICANCE: The use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.

Prevalence of keratinophilic fungi in soils of St. Kitts and Nevis.

INTRODUCTION: Information on the prevalence of keratinophilic fungi in West Indies is scanty. Occurrence of keratinophilic fungi in soils of St. Kitts and Nevis has not been investigated previously. METHODOLOGY: The prevalence of keratinophilic fungi was investigated in 108 samples of soils of varying habitats from St. Kitts and 55 such samples from Nevis by hair-baiting technique. Fungal growths appearing on the hair baits after four to eight weeks of incubation at room temperature were microscopically examined and cultured on mycological media. Cultures were identified on the basis of colonial and microscopic features. RESULTS: Forty-nine (45%) of the samples from St. Kitts and 38 (69%) from Nevis were positive for keratinophilic fungi. Microsporum gypseum complex, a well-known geophilic dermatophyte, was the most frequently recovered species being present in 15.7%  of soils of St. Kitts and 40% of soils of Nevis. The next commonest species recovered was Chrysosporium indicum, represented by 15 (13.9%) isolates from St. Kitts and seven (12.7%) isolates from Nevis. Other infrequently isolated keratinophilic species included Chrysosporium tropicum, Chrysosporium keratinophilum, and unidentified Chrysosporium species. CONCLUSIONS: This study is the first of its kind in the islands of St. Kitts and Nevis. A high incidence of M. gypseum complex in the soil of these islands is a noteworthy finding of public health significance.