Theresa Casey

Higher Stromal Expression of Transforming Growth Factor-beta Type II Receptors is Associated with Poorer Prognosis Breast Tumors

Transforming growth factor-beta (TGFB) is a potent inhibitor of normal epithelial cell proliferation, and may be one of the regulatory factors that are perturbed during tumor development. While many tumor cell lines no longer respond to the inhibitory effects of TGFB due to a reduction or absence of the type II receptor (TGFBR2), the role of TGFBR2 in tumors from patients with breast cancer is less clear. The objective of this study was to screen human breast tumors to determine if there was a TGFBR2 mutation and/or altered expression of TGFBR2 protein. Using 10 unique primers, SSCP-PCR was used to detect heterozygosity in the complete coding sequence from 72 tumors and normal DNA from 20 individuals. One region of the promoter was also examined. Expression of TGFBR2 in the same breast tumors was examined by immunohistochemistry. Sequence variations were identified among normal and tumor tissue samples by SSCP-PCR within coding regions of exon 4 (1/72 samples) and within non-coding regions of intron 2 (1/72), intron 3 (72/72), and intron 6 (1/72). A new polymorphism was identified in intron 3. Observed allele frequencies were consistent with Hardy–Weinberg equilibrium in both the tumors and normal DNA. TGFBR2 was expressed in the epithelium and stroma of tumor tissue. The percentage of cells expressing TGFBR2 in stroma was higher in patients that had a positive lymph node status and/or negative estrogen and progesterone receptor expression. There was no relationship between TGFBR2 expression in the epithelium and these variables.

Mammary epithelial cells treated concurrently with TGF-α and TGF-β exhibit enhanced proliferation and death

Transforming growth factor-α (TGF-α) stimulates while TGF-β inhibits mammary epithelial cell growth, suggesting that when cells are treated concurrently with the growth factors their combined effects would result in no net growth. However, combined treatments stimulate proliferation and cellular transformation in several cell lines. The objective of this paper was to describe the effect of long-term (6 days) concurrent TGF-α and TGF-β treatment on normal mammary epithelial cell growth pattern, morphology, and gene expression. Growth curve analysis showed that TGF-α enhanced while TGF-β suppressed growth rate until Day 4, when cells entered lag phase. However, cells treated concurrently with both growth factors exhibited a dichotomous pattern of growth marked by growth and death phases (with no intermittent lag phase). These changes in growth patterns were due to a marked induction of cell death from Day 2 (16.5%) to Day 4 (89.5%), resulting in the transition from growth to death phases, even though the combined treated cultures had significantly more (P < 0.05) cells in S phase on Day 4. TGF-β stimulated epithelial to mesenchyme transdifferentiation (EMT) in the presence of TGF-α, as characterized by increased expression of fibronectin and changes in TGF-β receptor binding. Expression patterns of genes that regulate the cell cycle showed significant interaction between treatment and days, with TGF-β overriding TGF-α–stimulated effects on gene expression. Overall, the combined treatments were marked by enhanced rates of cellular proliferation, death, and trans-differentiation, behaviors reminiscent of breast tumors, and thus this system may serve as a good model to study breast tumorigenesis.

INVOLUTION OF MOUSE MAMMARY GLANDS DURING WHOLE ORGAN CULTURE OCCURS VIA APOPTOSIS OF EPITHELIAL TISSUE

Apoptosis was measured in mammary glands during whole organ culture, to determine whether regression resulting from hormone withdrawal results in epithelial cell death as in vivo involution. Glands were evaluated for morphology and DNA degradation prior to whole organ culture, after lobulo‐alveolar development and 2, 4, or 6 days after hormone withdrawal. The data indicated that mammary regression during whole organ culture mimics in vivo involution and results in part from apoptosis of epithelial tissue.

Integration of a gene marker into mouse mammary glands during whole organ culture

The objective of this study was to develop a method to indelibly mark mammary cells to follow cell lineage. This was done by infecting mouse mammary glands in whole organ culture (WOC) with a retroviral vector containing the β-galactosidase (BAG) gene. Abdominal mammary glands from BALB/c mice primed with estrogen and progesterone were removed and injected with 30 µl of concentrated vector prepared from CRE BAG 2 (ATCC 1858 — CRL) cells or left uninjected and co-cultured with CRE BAG 2 feeder layers for 48 or 96 hours. Glands were floated on siliconized lens paper and incubated in Waymouths 752/1 medium supplemented with insulin, aldosterone, hydrocortisone, prolactin, and epidermal growth factor for 5 days. Integration of BAG was determined with a colorimetric assay using X-gal, a β-galactoside analog, in whole mounts and collagenase digested glands. Blue cells in collagenase digests and blue areas in whole mounts indicated that BAG had integrated into mammary cells during whole organ culture.