Keratins as markers that distinguish normal and tumor-derived mammary epithelial cells.

Abstract

Keratin 5 (K5) mRNA and protein are shown to be expressed in normal mammary epithelial cells in culture and are absent from tumor-derived cell lines. To extend these fridings, the full complements of keratins in normal, immortalized, and tumor cells were compared. It is shown here that normal cells produce keratins K5, K6, K7, K14, and K17, whereas tumor cells produce mainly keratins K8, K18, and K19. In immortalized cells, which are preneoplastic or partially transformed, the levels of K5 mRNA and protein are lower than in normal cells, whereas the amount of K18 is increased. Thus, K5 is an important marker in the tumorigenic process, distinguishing normal from tumor cells, and decreased K5 expression correlates with tumorigenic progression. (2, 4-11, 35, 36). Initial studies with keratins extracted from mammary glands showed differences between normal and tumor tissues (2).Immunohistochemical characterization has demonstrated that within the normal duct, basal and lumenal cells can be discriminated by the keratins they express. In these studies, basal cells, lying between the lumenal cells and the basement membrane, were characterized by expression of keratins K5 and K14, which are typical of myoepithelial cells in stratified epithelium, and lumenal cells were characterized by expression of simple epithelial keratins K8, K18, and K19 (12-15).These studies were performed in situ. Keratin expression may not be maintained after disruption of tissue architecture in cell culture. Medium constituents such as vitamin A, cAMP-elevating agents, epidermal growth factor, and other factors are known to affect keratin production (13,(16)(17)(18)37). The development of a medium capable of supporting the growth ofboth tumor and normal breast epithelial cells (1) has allowed us to make a comparison of their keratin profiles independent of medium effects. We have analyzed K5 as a potential marker for normal cells and have reviewed the array of keratins produced by cultured tumor and normal cells. cDNA Library Production. Total RNA was isolated from 184 cells by lysis with guanidinium isothiocyanate, centrifugation over a 5.7 M CsCl cushion, and purification of the resulting RNA pellet (24). Poly(A)+ RNA was purified by oligo(dT) chromatography using standard protocols (ref. 25, pp. 197-198). cDNA was made using the reverse transcriptase of Moloney murine leukemia virus (BRL) according to the supplier's instructions. cDNAs were blunt ended with T4 DNA polymerase, EcoRI linkers were ligated onto the cDNA, and the cDNA was cloned in the EcoRI site of AgtlO. MATERIALS AND METHODSSubtraction and Screening. The first-strand cDNA of 184 mRNA was hybridized with a 5-fold mass excess of 184B5KSVTu2 poly(A)+ RNA to Rot = 6000 mol (of nucleotide)-sec. Nonhybridizing nucleic acid was separated from DNARNA hybrids by chromatography over hydroxylapatite (26) and subjected to a further round of hybridization and hydroxylapatite chromatography. The final product (the "subtracted probe") was labeled by the random primer method (27)...

Analysis of polypeptide expression in benign and malignant human breast lesions: down-regulation of cytokeratins

“…We found cytokeratins 7,8,15 and 18 to be expressed in fibroadenomas. This result suggests that alterations in cytokeratin expression occur early during neoplastic transformation and is consistent with the finding of low levels of cytokeratin 5 and high levels of cytokeratin 18 in immortalised breast cell lines (Trask et al, 1990). The levels of cytokeratin 7,8,15 and 18 were significantly lower in carcinomas than in fibroadenomas.…”

Stem Cells in the Human Breast

“…An unexpected characteristic of these and other subsequently spontaneously immortalized human breast epithelial cell lines, as well as their transformed derivatives, is the conspicuous lack of a complete keratin K19-positive lineage (Trask et al 1990;Santner et al 2001). Therefore, irrespective of the fact that these cell lines have many properties in common with the mouse equivalent, COMMA-1D, in terms of stem cell properties, they fail to show the same broadness of differentiation repertoire.…”

Discovery of differentially expressed genes in human breast cancer using subtracted cDNA libraries and cDNA microarrays

“…We next performed DNA sequencing analysis of these 96 clones, which revealed several genes that are known to be overexpressed in breast tumors (Table 1). These genes are mammaglobin (Watson and Fleming, 1996), keratin (Trask et al, 1990), and estrogen receptor (Kurebayashi et al, 2000). Of the 96 clones examined, four clones did not match any entry (550% identity) in the public databases including GenBank DNA and human EST.…”

“…Sixty-two clones with a ratio between these two probe groups (all breast tumors vs all normal non-breast tissues) greater than three were selected and sequenced, as shown in Table 2. Among these clones there were 31 known genes including mammaglobin, 40 kDa keratin, and ®bronectin, which have previously been shown to be associated with breast cancers (Watson and Fleming, 1996;Loridon-Rosa et al, 1990;Trask et al, 1990).…”