Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

Multiple labeling techniques for fluorescence microscopy.

As described in Chapter 2 by Brooks, it has long been possible to localize antigens immunocytochemically using specific antibodies in conjunction with a label that is visible microscopically. Although much information can be derived by localizing a single protein/peptide, it is often useful to label simultaneously for two or more antigens within the same cells or tissue sections. There are a number of occasions when such multiple labeling techniques can be used: (1) to phenotype cells, for which no specific marker is available, using an appropriate panel of antibodies; (2) to identify which cells in a tissue or culture express an antigen of interest, by simultaneously labeling with antibodies to both this antigen and to a known phenotype marker; (3) to identify the distribution of an antigen at the subcellular level by simultaneously labeling with antibodies to both this and a known organelle marker; (4) to investigate whether several antigens of interest are colocalized, either at the cellular or the subcellular level. Although it is possible to directly label a primary antibody with a fluorochrome (direct immunofluorescence), the overall fluorescence signal achieved using this technique is often weak (2). Indirect immunofluorescence involves the use of secondary antibodies conjugated to different fluorochromes (2). This approach has the advantage that multiple secondary antibodies can bind to each primary antibody, resulting in an amplification of the signal. The most basic form of multiple labeling involves the simultaneous use of two or more primary antibodies that have been raised in different species of animals.

Proliferation and differentiation in the human breast during pregnancy.

Using multiple immunofluorescence labelling on human breast tissues obtained and freshly frozen at the 12th, 15th, and 18th weeks of pregnancy, we have shown that markers of mammary functional differentiation, milk proteins (beta-casein and kappa-casein), are synthesised by actively cycling (Ki67 positive) as well as non-cycling (Ki67 negative) cells. These results demonstrate that functional differentiation/maturation does not coincide with loss of proliferative potential in human mammary luminal epithelial cells. In addition, we have examined expression patterns of integrin subunits (alpha1, alpha2, alpha3, alpha6, beta1, and beta4) and extracellular matrix components (laminin, fibronectin, collagen I, and collagen IV), since they have been shown to exert influences on mammary differentiation and morphogenesis in vitro. Compared to human breast tissues obtained from non-pregnant women, a decrease in alpha2 labelling on luminal epithelial cells was observed, particularly in expanding acini that showed abundant Ki67 positivity. The expression patterns of other integrin subunits, however, did not change, indicating that the expression patterns of most integrins existing prior to pregnancy are sufficient to support the morphological and functional development associated with milk protein synthesis.

Differential expression of type XIV collagen/undulin by human mammary gland intralobular and interlobular fibroblasts.

Immunolocalisation of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma. The interlobular stroma is located between the breast lobules and their associated intralobular stroma. Fibroblasts isolated from the interlobular stroma synthesised 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesised type I and type IV collagens in similar amounts. The differential expression of type XIV collagen/undulin was maintained with passage in culture. The results suggest a role for type XIV collagen/undulin in stabilising dense collagen fibrils. The maintenance of two types of structurally distinct stromas may be important during developmental processes in the mammary gland.

Ectoenzyme regulation by phenotypically distinct fibroblast sub-populations isolated from the human mammary gland.

Inter- and intralobular mammary fibroblasts have been separated from normal human breast tissue and cultured to study the differential expression of ectoenzymes present within the stroma of the normal gland and associated with breast cancers. Specific ectoenzymes were identified by indirect immunofluorescence and quantified by flow cytometry and semi-quantitative PCR. A consistent difference was noted between the two fibroblast sub-populations at early passage in respect of dipeptidyl peptidase IV (DPP IV) and aminopeptidase N (APN) expression. Early passage intralobular fibroblasts were positive for APN but negative for DPP IV, as seen in the intact tissue. However, with continued sub-culture they gradually began to express DPP IV, until at later passages they became indistinguishable from the interlobular fibroblasts, which were APN and DPP IV-positive at all stages in culture, as they are in intact tissue. Neutral endopeptidase (NEP/CALLA/CD10) is not expressed by normal adult breast fibroblasts but is found in the stroma associated with over 60% of breast cancers. It was up-regulated in vitro on both inter- and intralobular fibroblasts, with final levels that were significantly (< 14 times) higher on the former in all pairs of preparations from individual donors analysed. This difference persisted with continued passage, and levels of the ectoenzyme and its messenger RNA were further up-regulated by hydrocortisone in both populations. These results demonstrate that phenotypically distinct cultures of human mammary fibroblast sub-populations can be used to study the regulation of these stromal ectoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalisation of cell surface peptidases in the developing human breast.

The immunocytochemical distribution of three cell surface peptidases was investigated in samples of developing infant breast ranging in age from newborn to 9.5 months. We have previously demonstrated that in the adult breast these enzymes identify subpopulations of epithelial cells and fibroblasts. We therefore wished to address two questions: (a) At what stage in breast development can fibroblast subpopulations be identified, and (b) Is the distribution of these peptidases related to cellular differentiation and morphogenesis? At the histological level there was a cuff of stromal cells closely associated with the developing ductular and lobular structures. At all stages of ductular and lobular development the fibroblasts in this layer were consistently negative for dipeptidyl peptidase IV (DPP IV) and clearly distinguished from the fibroblasts in the surrounding matrix, some of which expressed DPP IV in an age-dependent manner. Within the infant breast aminopeptidase N (APN) was localised to luminal epithelial cells and all fibroblasts, whilst neutral endopeptidase (NEP) was specifically localised to myoepithelial cells. These results are considered in relation to the role of stromal-epithelial interactions during morphogenesis and the proposed function of these enzymes.

An appraisal of low-temperature embedding by progressive lowering of temperature into Lowicryl HM20 for immunocytochemical studies.

The progressive lowering of temperature (PLT) method of embedding for electron microscope immunolabelling has been examined with the objective of formulating a standardized protocol which can be applied to a wide variety of samples. The methods described cover fixation, processing of samples by the PLT method, embedding in Lowicryl HM20 and subsequent immunolabelling. Each of the steps in the fixation and embedding protocol has been assessed for its potential to retain both morphology and antigenicity. Comparison of samples embedded in Lowicryl K4M and HM20 at -25 degrees C indicate an increased membrane contrast in HM20 sections, and a further improvement in morphology when samples were embedded in HM20 at -50 degrees C rather than at -25 degrees C. The results of applying the methods described are demonstrated in a range of samples of both mammalian and botanical origin, which includes solid tissues, cells in suspension, and cells grown in vitro as a monolayer culture and embedded in situ. Samples processed by this method have been immunolabelled using a wide range of antibodies recognizing nuclear, cytoplasmic, cell membrane and extracellular matrix antigens.

Immunocytochemical localization of the ectoenzyme aminopeptidase N in the human breast.

The ectoenzyme aminopeptidase N (APN) was localized in the normal human breast at both the light microscopic and the ultrastructural level. APN was expressed on intralobular and interlobular fibroblasts and on the apical surface of some luminal epithelial cells. This enzyme was not detected on either myoepithelial cells and their associated basement membrane or capillary endothelium. Furthermore, the staining pattern was maintained in benign and malignant breast disease. APN belongs to a family of enzymes that hydrolyze peptides in the extracellular space. As with other ectoenzymes present in the breast, APN expression is restricted to specific cell types. This pattern of expression may indicate a role for these enzymes in the biology of the normal breast.

Dipeptidyl peptidase IV expression identifies a functional sub-population of breast fibroblasts.

The immunocytochemical distribution of the cell-surface enzyme dipeptidyl peptidase IV (DPP IV) has been studied in the human breast at the light and ultrastructural level. The presence of the enzyme was demonstrated on the cell membranes of interlobular fibroblasts, whilst intralobular fibroblasts were DPP-IV-negative. A fluorograph, after immunoprecipitation of 35S-methionine-labelled proteins of fibroblasts from primary breast cultures with an anti-serum to DPP IV, demonstrated a band at 135 kDa consistent with the presence of the enzyme. The clear delineation of 2 functionally distinct subpopulations of breast fibroblasts was maintained in benign fibro-adenomas and cystosarcoma phyllodes, both tumour types having growth characteristics of intralobular stroma. This observation has important implications for both normal breast biology and for breast carcinogenesis.