Gap junction distribution and connexin expression in human breast.

Expression of the gap junction proteins (connexins) in human breast epithelium was studied in vivo and in vitro. A panel of sequence-specific anti-peptide antibodies was used to examine four connexin (Cx) isoforms by indirect immunofluorescence labeling. Antibodies to Cx43 readily detected gap junctions between the basal cells in major ducts, but less so within lobular/alveolar structures. Cx26 immunoreactivity was less abundant in beast epithelium but was observed between the luminal cells in major ducts and to a lesser extent in lobular/alveolar structures. Ultrastructural studies of normal human breast showed gap junctions between basal cells in ducts and lobules, but not between luminal cells or between luminal and basal cells. Immunomagnetically separated luminal and basal cells were grown in vitro. Basal cells expressed Cx43 at cell-cell attachment points whereas luminal cells showed only small amounts of immunolabeling with the Cx26 antibody which was generally not associated with the cell borders. Microinjection of Lucifer yellow into cultured luminal or basal cells indicated that basal cells have high levels of gap junctional communication, but dye transfer between luminal cells was difficult to detect by transfer of Lucifer yellow. Western blot analysis of purified luminal and basal cells indicated the presence of mainly Cx43 in both cell types. Polymerase chain reaction analysis of breast mRNA identified message for CX43 and to a lesser extent for Cx26; Cx32 was not detected in human breast, although it was present in mouse mammary gland. mRNA extracted from cloned cultures of human luminal and basal cells contained message for Cx43 and Cx26 in both cell types.

Epidermal growth factor receptor expression on human breast luminal and basal cells in vitro.

The expression of EGF receptors has been studied on luminal and basal cells of human breast in vitro. Primary cultures of normal adult human breast epithelium were prepared as single cell suspensions containing a mixture of luminal and basal cells. The cells were simultaneously immunolabelled with antibodies recognising EMA (luminal epithelial cells), CALLA/CD10 (basal cells) and the epidermal growth factor receptor (EGFR). Flow cytometric analysis of these triple labelled cells detected low levels of EGFR on both cell types, with proportionally more EGFR on basal cells compared with luminal cells. Separated populations of basal and luminal cells were prepared from single cell suspensions by flow sorting or by immunomagnetic methods and cultured with and without EGF. Increased proliferation was detected in both cell types in the presence of EGF. To determine the localisation of the EGF receptor, purified cell populations were immunolabelled with anti-EGFR antibody and an FITC-labelled second antibody for fluorescence light microscopy and colloidal gold-labelled antibody for scanning electron microscopy (SEM). Low levels of EGFR were detected by indirect immunofluorescence on both cell types with higher levels on basal cells compared with luminal cells. The detailed subcellular distribution of the receptor was examined by SEM, with gold-labelling of EGFR detected using a field emission scanning electron microscope with a YAG crystal backscattered electron detector. Both luminal and basal cells expressed EGFR over the upper surface of individual cells when these were growing in isolation, but when cells formed part of a confluent island, levels of EGFR on the upper surface of cells were obviously reduced. Observations made by SEM on cells at the edges of such confluent islands showed that cultured basal cells expressed much higher levels of EGFR on their basal, as compared with their upper surfaces.

Effects of growth factors on proliferation on basal and luminal cells in human breast epithelial explants in serum-free culture.

A method of culturing human breast epithelium is described in which viable explants can be maintained in protein-free medium while retaining the capacity of responding to added hormones and growth factors for at least 7 days. Culture parameters were chosen to provide maximum sensitivity of detection of proliferative responses by autoradiography. Under basal conditions, the mean thymidine labeling index of the explants was 0.08%. After stimulation with insulin, hydrocortisone, and cholera toxin (I,H,CT), a combination known to stimulate proliferation in human breast epithelium in vitro, the mean labeling index was 15.7%. Stimulation of explants with epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha resulted in mean labeling indices of 6.6 and 10.8%, respectively. Autoradiography at the ultrastructural level demonstrated that in I,H,CT-stimulated explants the majority of the labeled cells were luminal, with only 1.5% being basal cells. In contrast, after EGF and TGF-alpha basal cells accounted for 11.5 and 18.5% of the labeled population. These results indicate that this system provides an in vitro assay of proliferative activity in the normal human breast that enables comparisons to be made between both the luminal and the basal cells in the explants and their counterparts in monolayer culture prepared from flow sorted cells. Thus, growth responses dependent on cell-to-cell interactions or stromal modulation can be identified.

Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture.

Normal human breast explants were maintained in serum-free culture for 7 days in the presence of either insulin hydrocortisone and cholera toxin (I/H/CT), epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). Explants were labelled with [3H] thymidine, fixed in methacarn and processed for autoradiography. Parallel sections were immunolabelled with anti-PCNA antibody and analysed with a CAS 200 image analyser. Thymidine labelling index (TLI) and PCNA expression produced similar results with both indices increased in response to I/H/CT, EGF and TGF-alpha. In sections double labelled for PCNA and autoradiography the majority of labelled cells were positive for both markers.

Peripubertal human breast development.

The microanatomical and histological appearance of the human breast has been studied during puberty. The macroscopic architecture of the mammary epithelial tree was identified and correlated with the histological appearance of material excised from defined regions of the breast preparations. Between ages 13 yrs and 15 yrs the human breast shows evidence of ductal elongation and branching, with lobules formed by lateral and dichotomous branching. The majority of ducts are lined by a two-layered epithelium consisting of recognisable myoepithelial and luminal cells. Less-well-defined multilayered regions were also observed in some areas, apparently at the site of lateral branching or early lobular development.