2D Difference Gel Electrophoresis Analysis of Different Time Points during the Course of Neoplastic Transformation of Human Mammary Epithelial Cells

Abstract

Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesis over an 80 day period, protein extracts were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). Nine proteins were found to be significantly different in abundance: keratin 1, keratin 7, heat shock protein 4A-like, t-complex protein 1, stathmin, gelsolin, FK506 binding protein 5, ribosomal protein P0, and maspin. Keratin 7 and maspin displayed a linear down-regulation over 80 days. All of these proteins have been shown to be involved in the maintenance of a metastatic state including cytoskeletal modifications and motility. We conclude that, following neoplastic induction, THMECs display an early and progressive increase in metastatic potential. Further investigations into the function and regulatory mechanisms of these proteins will provide an unparalleled understanding of the initial states through which a breast cancer cell transitions following acquisition of the genetic abnormalities required for oncogenesis.

Epigenetic Regulation of Multiple Tumor-Related Genes Leads to Suppression of Breast Tumorigenesis by Dietary Genistein

“…In current study, we introduced a well studied cellular cancer transformation system that closely mimics the process of early human breast tumorigenesis since we have extensive experience using this model for cancer research [27], [32], [33]. In this model, three defined genetic elements including SV40, hTERT and H-RAS were introduced into normal HMECs (Fig.…”

“…This resulted in a neoplastic transformation and generation of different stages of human mammary cancer cells, including precancerous SH cells (transfected with SV40 and hTERT ) and transformed breast cancer SHR cells (transfected with all three genetic elements). This system has been verified for its successive genetic transfection and carcinogenic transformation by various experiments including protein expression, anchorage-independent colony formation and tumorigenesis in immunedeficient nude mice [26], [27]. Cell clones for precancerous SH cells and transformed breast cancer SHR cells used in this study were established in our laboratory at around 20 population doublings when experiments were initiated.…”

“…An established cancer chemoprevention model that causes normal breast cells to undergo cancer initiation was used in this study. This transformation model system was originally developed by Weinberg and his colleagues in 1999, which has been accomplished oncogenic transformation through the viral-mediated serial gene transfer of three defined elements, SV40, hTERT and hRAS-V12 , to normal human epithelial cells [25], [26] Although these individual genetic mutations are not universally found in all types of breast cancer, the model has been widely used for breast cancer research since it can closely approximate the initiation and early progression of breast cancer [27], [42]. We therefore feel that this model enables us to assess the impact of the GE in real-time not only in preventing the transition of oncogenesis, but in preventing the early epigenetic aberrations that are frequently associated with cancer.…”

“…Early transformed SH cells (precancerous cells; normal HMECs stably transfected with SV40 and human telomerase reverse transcriptase, hTERT ) and completely transformed SHR cells (breast cancer cells; SH cells except with added H-Ras ) were established in our laboratory (Fig. 1A) [27]. An established breast cancer cell line, MDA-MB-231 cells, obtained from the American Type Culture Collection (ATCC) was used as a reference.…”

Docosahexaenoic acid inhibits the invasion of MDA-MB-231 breast cancer cells through upregulation of cytokeratin-1

“…in addition, Krt1 was shown to interact with the tyrosine kinase Src through binding to integrin β1 (23) and therefore the presence of Krt1 in a crude membrane preparation is not surprising. it has been shown that Krt1 level is strongly decreased in breast cancer cells reaching a metastatic phenotype (40). in addition, a recent study has shown that Krt1 is decreased in breast tumors (41).…”