Bernard W Futscher

Desmocollin 3 (DSC3) is a member of the cadherin superfamily of calcium-dependent cell adhesion molecules and a principle component of desmosomes. Desmosomal proteins such as DSC3 are integral to the maintenance of tissue architecture and the loss of these components leads to a lack of adhesion and a gain of cellular mobility. DSC3 expression is down-regulated in breast cancer cell lines and primary breast tumors; however, the loss of DSC3 is not due to gene deletion or gross rearrangement of the gene. In this study, we examined the prevalence of epigenetic silencing of DSC3 gene expression in primary breast tumor specimens.

PMID: 2534817;Abstract:

In this report we present evidence which suggests that pretreatment of a highly nitrosourea-resistant human colon tumor cell line with non-cytotoxic doses of streptozotocin (STZ) prior to, or simultaneously with, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) exposure produces synergistic increases in cytotoxicity of several logs over the cytotoxicity produced by exposure to BCNU alone. STZ pretreatment or simultaneous treatment with BCNU allowed BCNU-induced DNA interstrand crosslinks to form in this cell line, in which BCNU alone did not induce DNA interstrand-crosslinks. Reversal of the schedule (i.e., STZ following BCNU) was less effective in producing synergistic cell kill or increased DNA interstrand crosslinking. Replacement of BCNU with fresh BCNU was not effective in producing increased cell kill and produced no observable interstrand crosslinking. Direct assays for guanine O6-DNA alkyltransferase activity confirmed that more than 75% inhibition of this important DNA repair system occurred following exposure to 2.5 mM STZ and that the inhibition was virtually complete when STZ pretreatment was combined with BCNU exposure.

PMID: 12021783;Abstract:

The nucleotide 5-methylcytosine is involved in processes crucial in mammalian development, such as X-chromosome inactivation and gene imprinting. In addition, cytosine methylation has long been speculated to be involved in the establishment and maintenance of cell type-specific expression of developmentally regulated genes; however, it has been difficult to identify clear examples of such genes, particularly in humans. Here we provide evidence that cytosine methylation of the maspin gene (SERPINB5) promoter controls, in part, normal cell type-specific SERPINB5 expression. In normal cells expressing SERPINB5, the SERPINB5 promoter is unmethylated and the promoter region has acetylated histones and an accessible chromatin structure. By contrast, normal cells that do not express SERPINB5 have a completely methylated SERPINB5 promoter with hypoacetylated histones, an inaccessible chromatin structure and a transcriptional repression that is relieved by inhibition of DNA methylation. These findings indicate that cytosine methylation is important in the establishment and maintenance of cell type-restricted gene expression.

PMID: 9271436;PMCID: PMC232409;Abstract:

O⁶-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O⁶-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O⁶- alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line. RPMI g226/S, and its MGMT-negative variant termed 8226N, to study the control of MGMT expression. The loss of MGMT activity in 8226N was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.

PMID: 8006031;Abstract:

Expression of the O⁶-methylguanine DNA methyl-transferase (MGMT) gene in human glioma cell lines is strongly associated with resistance to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea. To examine the possibility that methylation of the body and promoter regions of the MGMT gene is associated with MGMT expression in a graded, rather than a completely on/off fashion, the present study analyzed the methylation status of the MGMT gene in human glioma cell lines exhibiting a wide range of MGMT expression. Methylation in the body of the gene was uniform within each cell line and correlated directly with MGMT expression. The level of MGMT promoter methylation was also graded across the cell lines, at 21 of 25 CpGs tested, but correlated inversely with MGMT expression. Two sites in the MGMT promoter were also much more accessible to restriction enzyme digestion, and thus in a more open chromatin conformation, in nuclei from high MGMT expressors relative to nuclei from cells with little or no MGMT expression. We conclude that the level of methylation, in both the body and promoter of the MGMT gene, is associated with MGMT expression in a graded fashion and may be important in setting the transcriptional state of the MGMT promoter through changes in chromatin structure.