Bernard W Futscher

PMID: 9778046;Abstract:

BRCA1 mRNA is reduced in sporadic breast cancer cells despite the lack of mutations. Because a CpG island is found at the 5' end of the BRCA1 gene, we hypothesized that the decreased BRCA1 mRNA in sporadic breast cancer was associated with aberrant cytosine methylation of the CpG island. We examined BRCA1 mRNA expression in normal human mammary epithelial cells (HMECs), peripheral blood lymphocytes (PBLs) and six sporadic breast cancer cell lines using RT-PCR. The normal breast cells expressed high levels of BRCA1 mRNA. The sporadic breast cancer cell lines and PBLs expressed lower levels of BRCA1 mRNA ranging from a 3-16-fold decrease compared to the normal breast cells. We identified a 600 bp region of the BRCA1 CpG island that possessed strong promoter activity (~40-fold above control), and determined the cytosine methylation patterns of the 30 CpG sites within this region by sodium bisulfite genomic sequencing. The HMECs, PBLs and five of the sporadic breast cancer cell lines mere largely unmethylated. However, one sporadic breast cancer cell line, UACC3199, was ≥ 60% methylated at all 30 CpG sites (18 sites were 100% methylated) and was associated with an eightfold decrease in BRCA1 mRNA compared to normal breast cells. These findings suggest that aberrant cytosine methylation of the BRCA1 CpG island promoter may be one mechanism of BRCA1 repression in sporadic breast cancer.

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

PMID: 18588940;PMCID: PMC2567114;Abstract:

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 μM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 μM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 μM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure. © 2008 Elsevier Ireland Ltd. All rights reserved.

PMID: 11359780;PMCID: PMC2824502;Abstract:

The α6 integrin is a 140-kDa (nonreduced) laminin receptor. We have identified a novel 70-kDa (nonreduced) form of the α6 integrin called α6p for the latin word parvus, meaning small. The variant was immunoprecipitated from human cells using four different α6-specific monoclonal antibodies but not with α3 or α5 antibodies. The α 6p integrin contained identical amino acid sequences within exons 13-25, corresponding to the extracellular "stalk region" and the cytoplasmic tail of the α6 integrin. The light chains of α6 and α6p were identical as judged by α6A-specific antibodies and electrophoretic properties. The α6p variant paired with either β1 or β4 subunits and was retained on the cell surface three times longer than α6. Reverse transcription/polymerase chain reaction analysis revealed a single polymerase chain reaction product. The α6p variant was found in human prostate (DU145H, LnCaP, PC3) and colon (SW480) cancer cell lines but not in normal prostate (PrEC), breast cancer (MCF-7), or lung cancer (H69) cell lines or a variant of a prostate carcinoma cell line (PC3-N). Protein levels of α6p increased 3-fold during calcium-induced terminal differentiation in a normal mouse keratinocyte model system. A novel form of the α6 integrin exists on cell surfaces that contains a dramatically altered extracellular domain.