Lee, J. K., Garbe, J. C., Vrba, L., Miyano, M., Futscher, B. W., Stampfer, M. R., & LaBarge, M. A. (2015). Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells. Frontiers in cell and developmental biology, 3, 13.
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.
Eblin, K. E., Hau, A. M., Jensen, T. J., Futscher, B. W., & Gandolfi, A. J. (2008). The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: Acute studies. Toxicology, 250(1), 47-54.
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.
Davis, T. L., Rabinovitz, I., Futscher, B. W., Schnölzer, M., Burger, F., Liu, Y., Kulesz-Martin, M., & Cress, A. E. (2001). Identification of a Novel Structural Variant of the α6 Integrin. Journal of Biological Chemistry, 276(28), 26099-26106.
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.
Domann, F. E., & Futscher, B. W. (2003). Editorial: Maspin as a molecular target for cancer therapy. Journal of Urology, 169(3), 1162-1164.
Futscher, B., Jensen, T. J., Novak, P., Eblin, K. E., Gandolfi, A. J., & Futscher, B. W. (2008). Epigenetic remodeling during arsenical-induced malignant transformation. Carcinogenesis, 29(8).
Humans are exposed to arsenicals through many routes with the most common being in drinking water. Exposure to arsenic has been associated with an increase in the incidence of cancer of the skin, lung and bladder. Although the relationship between exposure and carcinogenesis is well documented, the mechanisms by which arsenic participates in tumorigenesis are not fully elucidated. We evaluated the potential epigenetic component of arsenical action by assessing the histone acetylation state of 13 000 human gene promoters in a cell line model of arsenical-mediated malignant transformation. We show changes in histone H3 acetylation occur during arsenical-induced malignant transformation that are linked to the expression state of the associated gene. DNA hypermethylation was detected in hypoacetylated promoters in the select cases analyzed. These epigenetic changes occurred frequently in the same promoters whether the selection was performed with arsenite [As(III)] or with monomethylarsonous acid, suggesting that these promoters were targeted in a non-random fashion, and probably occur in regions important in arsenical-induced malignant transformation. Taken together, these data suggest that arsenicals may participate in tumorigenesis by altering the epigenetic terrain of select genes.