Bernard W Futscher

PMID: 23386909;PMCID: PMC3563072;Abstract:

Background: Inflammatory breast cancer (IBC) is a rare, highly aggressive form of breast cancer. The mechanism of IBC carcinogenesis remains unknown. We sought to evaluate potential genetic risk factors for IBC and whether or not the IBC cell lines SUM149 and SUM190 demonstrated evidence of viral infection. Methods: We performed single nucleotide polymorphism (SNP) genotyping for 2 variants of the ribonuclease (RNase) L gene that have been correlated with the risk of prostate cancer due to a possible viral etiology. We evaluated dose-response to treatment with interferon- alpha (IFN-a); and assayed for evidence of the putative human mammary tumor virus (HMTV, which has been implicated in IBC) in SUM149 cells. A bioinformatic analysis was performed to evaluate expression of RNase L in IBC and non-IBC. Results: 2 of 2 IBC cell lines were homozygous for RNase L common missense variants 462 and 541; whereas 2 of 10 non-IBC cell lines were homozygous positive for the 462 variant (p= 0.09) and 0 of 10 non-IBC cell lines were homozygous positive for the 541 variant (p = 0.015). Our real-time polymerase chain reaction (RT-PCR) and Southern blot analysis for sequences of HMTV revealed no evidence of the putative viral genome. Conclusion: We discovered 2 SNPs in the RNase L gene that were homozygously present in IBC cell lines. The 462 variant was absent in non-IBC lines. Our discovery of these SNPs present in IBC cell lines suggests a possible biomarker for risk of IBC. We found no evidence of HMTV in SUM149 cells. A query of a panel of human IBC and non-IBC samples showed no difference in RNase L expression. Further studies of the RNase L 462 and 541 variants in IBC tissues are warranted to validate our in vitro findings. ©Ivyspring International Publisher.

PMID: 8968098;Abstract:

A new human myeloma cell line, 8226/MDR10V, was selected from a P- glycoprotein-positive cell line, 8226/Dox40, in the continuous presence of doxorubicin and verapamil. MDR10V cells are 13-fold more resistant to doxorubicin and 4-fold more resistant to vincristine than the parent cell line, Dox40. Chemosensitizers are also less effective in reversing resistance in the MDR10V compared to the Dox40 cells. Despite higher resistance to cytotoxic agents, MDR10V expresses 40% less P-glycoprotein in the plasma membrane compared to Dox40; however, total cellular P- glycoprotein is the same in both cell lines. Confocal immunofluorescence microscopy shows 2.5-fold more P-glycoprotein in the cytoplasm of MDR10V cells as compared to Dox40 cells. The cytoplasmic location of P- glycoprotein in the MDR10V cells is associated with a redistribution of doxorubicin. In Dox40 cells, doxorubicin is concentrated in the nucleus, whereas in MDR10V cells, 90% of doxorubicin is found in the cytoplasm. In the presence of equivalent intracellular doxorubicin, there was a decrease in DNA-protein crosslinks in the MDR10V cell line compared to the Dox40 cell line. This finding is in agreement with the intracellular doxorubicin fluorescence studies showing less doxorubicin in the nuclei of MDR10V cells compared to Dox40 cells. Verapamil is less effective in increasing doxorubicin accumulation in the nuclei of MDR10V cells compared to Dox40 cells. Processing of P-glycoprotein from the endoplasmic reticulum to the medial Golgi was identical between the two cell lines as determined by endoglycosidase H sensitivity of newly sensitized P-glycoprotein. No mutations were found in MDR1 cDNA from MDR10V cells compared to Dox40 cells. These results suggest that resistance to chemosensitizing agents plus cytotoxic drugs is associated with a redistribution of P-glycoprotein from the plasma membrane to the cytoplasm, which in turn reduces the amount of cytotoxic drug reaching the nucleus.

miRNAs are important regulators of gene expression that are frequently deregulated in cancer, with aberrant DNA methylation being an epigenetic mechanism involved in this process. We previously identified miRNA promoter regions active in normal mammary cell types and here we analyzed which of these promoters are targets of aberrant DNA methylation in human breast cancer cell lines and breast tumor specimens. Using 5-methylcytosine immunoprecipitation coupled to miRNA tiling microarray hybridization, we performed comprehensive evaluation of DNA methylation of miRNA gene promoters in breast cancer. We found almost one third (55/167) of miRNA promoters were targets for aberrant methylation in breast cancer cell lines. Breast tumor specimens displayed DNA methylation of majority of these miRNA promoters, indicating that these changes in DNA methylation might be clinically relevant. Aberrantly methylated miRNA promoters were, similar to protein coding genes, enriched for promoters targeted by polycomb in normal cells. Detailed analysis of selected miRNA promoters revealed decreased expression of miRNA linked to increased promoter methylation for mir-31, mir-130a, let-7a-3/let-7b, mir-155, mir-137 and mir-34b/mir-34c genes. The proportion of miRNA promoters we found aberrantly methylated in breast cancer is several fold larger than that observed for protein coding genes, indicating an important role of DNA methylation in miRNA deregulation in cancer.

PMID: 1740017;Abstract:

We initiated this study to determine whether three structurally related bifunctional alkylating agents could induce the expression of a presumptive human DNA repair gene. The gene chosen for this study is known to encode the ribosomal phosphoprotein PO, but ironically may also share functions related to DNA repair. We now show by Northern analysis that PO is induced by L-phenylalanine mustard, 4-hydroperoxycyclophosphamide and mechlorethamine, which are DNA-damaging agents commonly used as chemotherapeutic antitumor agents. In further support of its involvement in DNA repair is the finding of a 30- to 50-fold constitutive overexpression of the PO gene in human tumor cell lines that are Mer^-, cells which lack O⁶-methylguanine methyltransferase activity, when compared to Mer⁺ cell lines. This constitutively elevated level of PO in Mer^- cell lines, which are thus DNA repair defective for O⁶-alkyguanine lesions, was not observed for other genes tested, including the human ribosomal gene S17 whose mRNA steady-state levels were uniformly the same in both Mer^- and Mer⁺ cells. Taking these data together, it appears that increased levels of PO are somehow linked to DNA repair, and increased expression of PO may compensate for the decreased O⁶-methylguanine DNA methyltransferase activity in Mer^- cells. Furthermore, the PO gene has also been shown to be overexpressed in colorectal tumors and polyps and the sera of some systemic lupus erythematosus patients contain antibodies against PO. The titer of the anti-PO antibodies rises significantly during lupus psychosis.

PMID: 19509227;PMCID: PMC2697259;Abstract:

The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16^INK4A expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment. ©2009 American Association for Cancer Research.