Howlett A R, Petersen O W, Steeg P S, Bissell M J
Division of Life Sciences, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
J Natl Cancer Inst. 1994 Dec 21;86(24):1838-44. doi: 10.1093/jnci/86.24.1838.
We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia.
Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation.
The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest.
In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship.
The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene expression and morphological-biosynthetic-growth aspects of breast differentiation in this model system.
While the basement membrane microenvironment is capable of directing the differentiation of normal human breast cells, neoplastic transformation abrogates this relationship, suggesting that intrinsic cellular events are also critical to this process. The data identify nm23-H1 gene expression as one of these events, suggesting an important role in the modulation of cellular responsiveness to the microenvironment. The data also identify previously unknown growth inhibitory effects of nm23-H1 gene overexpression.
我们开发了一种使用重组基底膜成分的培养系统，在该系统中，正常人类乳腺上皮细胞展现出发育和分化过程的多个方面，包括腺泡样结构的形成、基底膜成分的产生和基底沉积，以及唾液粘蛋白的产生和顶端分泌。来自人类乳腺癌的细胞系和培养物无法重现这一过程。数据表明细胞与基底膜的相互作用在正常乳腺分化调节中具有重要性，并且在肿瘤形成过程中可能会丧失这种相互作用。
我们的目的是利用该检测方法研究假定的转移抑制基因nm23-H1在乳腺发育和分化中的作用。
将转移性人类乳腺癌细胞系MDA-MB-435、用对照pCMVBamneo载体转染的克隆，以及用含有nm23-H1互补DNA的pCMVBamneo载体转染的克隆（后者在体内自发转移潜能显著降低）在重组基底膜中培养。检测克隆形成腺泡样球体、基底膜成分沉积、唾液粘蛋白产生、极化和生长停滞的情况。
与亲代细胞系和对照转染子相比，过表达Nm23-H1蛋白的MDA-MB-435乳腺癌细胞在重组基底膜中恢复了正常表型的多个方面。Nm23-H1蛋白阳性细胞形成有组织的腺泡样球体，在球体外部沉积IV型胶原和一定程度的层粘连蛋白等基底膜成分，表达唾液粘蛋白，并生长停滞。Nm23-H1蛋白阳性细胞的生长停滞发生在基底膜形成之前且与之相关，提示存在因果关系。
数据表明在该模型系统中，nm23-H1基因表达与乳腺分化的形态学-生物合成-生长方面之间存在一种先前未被识别的因果关系。
虽然基底膜微环境能够指导正常人类乳腺细胞的分化，但肿瘤转化消除了这种关系，这表明内在细胞事件对这一过程也至关重要。数据将nm23-H1基因表达确定为这些事件之一，表明其在调节细胞对微环境的反应性方面具有重要作用。数据还确定了nm23-H1基因过表达以前未知的生长抑制作用。
