Normal human breast epithelial cells show a Studies in the rodent have shown that the extracellular matrix regulates the growth and differentiation of normal mammary epithelial cells in vivo and in culture (6-13).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Thus far, efforts to study early events in human breast tumor development have been hampered by the lack of adequate means to distinguish between normal and transformed cells in culture and have relied on the criteria of senescence and immortalization (14-19)-phenomena that as yet do not have clear counterparts in vivo and take a considerable amount of time to establish.We asked whether normal human breast epithelial cells could respond to BM and recapitulate certain aspects of their normal growth and differentiation program as has been shown using rodent models (8-12). We show that human mammary epithelial cells can indeed express a normal pattern of growth and differentiation when cultured within a reconstituted BM derived from the Englebreth-Holm-Swarm (EHS) tumor. Furthermore, in response to exogenous BM, the cells basally deposit an endogenous BM. In contrast, carcinoma cell lines and biopsies from primary breast tumors were not capable of responding appropriately to BM nor were they able to deposit an intact endogenous BM.Other investigators have also used an EHS matrix to study human cell lines, including metastatic breast tumor cells (20)(21)(22)(23)(24)(25). These studies have focused on morphology of the cells on an EHS matrix. By including primary tissues and addressing growth as well as three-dimensional architecture in normal and malignant cell lines and primary cultures, we have been able to exploit cell-extracellular matrix interaction as an assay not only to distinguish between normal and malignant breast cells but also possibly to "grade" atypia and malignancy. In addition, the system can be used to define differentiation markers and to delineate early changes in transformation assays or preneoplastic lesions. MATERIALS AND METHODSCell Culture. Primary breast epithelia were prepared from 12 reduction mammoplasties and three breast carcinoma biopsies (two primaries and one lymph node metastasis). The primary specimens were selected, disaggregated, and cultured in serum-free CDM3 medium (26) without Hepes or phenol red in the basal medium (Dulbecco's modified Eagle's medium/F12) and with triiodothyronine at 10 nM. We used two normal breast epithelial cell lines and MCF-1OA (28)], another "normal" line [HBL-100 (29)], and six tumorigenic breast cell lines. These included HMT-3909S13 (30, 31), MCF-7 (including subline 9), ZR75, T47-D, and 33), and CAMA-1 (34). These were cultured as described initially except for MCF-10A, which was cul- tTo whom reprint requests should be sent at the * address.9064

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HGK (hepatocyte progenitor kinase-like/germinal center kinase-like kinase) is a member of the human STE20/mitogen-activated protein kinase kinase kinase kinase family of serine/threonine kinases and is the ortholog of mouse NIK (Nck-interacting kinase). We have cloned a novel splice variant of HGK from a human tumor line and have further identified a complex family of HGK splice variants. We showed HGK to be highly expressed in most tumor cell lines relative to normal tissue. An active role for this kinase in transformation was suggested by an inhibition of H-Ras V12 -induced focus formation by expression of inactive, dominantnegative mutants of HGK in both fibroblast and epithelial cell lines. Expression of an inactive mutant of HGK also inhibited the anchorage-independent growth of cells yet had no effect on proliferation in monolayer culture. Expression of HGK mutants modulated integrin receptor expression and had a striking effect on hepatocyte growth factor-stimulated epithelial cell invasion. Together, these results suggest an important role for HGK in cell transformation and invasiveness.The mammalian STE20/mitogen-activated protein kinase kinase kinase kinase (MAP4K) family consists of 28 serine/threonine kinases related in their catalytic domains (reviewed in reference 14). By analogy with the prototype STE20 kinase in Saccharomyces cerevisiae, mammalian MAP4K kinases are likely to regulate changes in transcription, cytoskeletal organization, and cell cycle progression in response to extracellular signals (17). Comparison of the overall domain structure places these kinases into two structural classes, the p21-activated protein kinases (PAKs) (1) and germinal center kinases (GCKs) (28). The GCK kinases lack the regulatory Cdc42/ Rac-interacting domain found in the PAKs, having instead an N-terminal kinase domain and a C-terminal extension of variable length. Unlike PAKs, several GCKs appear to be activated in the absence of stimuli when overexpressed (3,10,38), suggesting that these kinases are regulated either by oligomerization or by binding of negative regulatory factors.GCK kinases show little sequence homology outside of the kinase domain and are further broken down into nine subfamilies (14, 31a). HGK (hepatocyte progenitor kinase-like/GCKlike kinase) is one of four members of the GCK group IV (recently renamed the MSN subfamily) that also includes TNIK, MINK, and NRK/NESK (13,22,27,35,51). HGK, TNIK, and MINK are highly homologous in their kinase and C-terminal domains (about 92 and 87% amino acid identity, respectively), with variability in the intervening region that is less conserved (53% between HGK and TNIK). NRK/NESK is more divergent, with only 59% homology in the kinase domain and 37% homology in the C-terminal domain. The C-terminal domain is a citron homology (CNH) domain, named for citron rho-interacting kinase (CRIK), where it was first described (16,31). CNH domains are found not only in the GCK group IV/MSN kinases but also in group I GCKs (KHS subfamily) (14, 31a), as well as in pr...

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