Reversion of the Malignant Phenotype of Human Breast Cells inThree-Dimensional Culture and In Vivo by Integrin BlockingAntibodies Page: 4 of 31
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cells, generated from passage 118, became spontaneously tumorigenic after an additional 120
passages (passage 238), while the parental line remained nontumorigenic for greater than 400
passages (Nielsen et al., 1994; Briand et al., 1996). In this study we used nonmalignant S-1 cells
at passage 50 ("normal"; S-1) and the tumorigenic progeny at passage 238 (T4-2; referred
to as mt-1 in Briand et al., 1996). The latter cells were shown to have a trisomy of chromosome
7p in addition to other genetic alterations such as a p53 mutation and a cmyc amplification
(Madsen et al., 1992; Moyret et al., 1994; Nielsen et al., 1994; Briand et al., 1996). These two
cell lines, one originating from the other by spontaneous genetic events, therefore, provide a
unique tool for addressing the specific mechanisms involved in malignant conversion in
the breast. In this paper, we postulated that if there were a cause and effect relationship between
perturbed tissue organization, loss of cell-cell interactions and altered ECM-signaling through
integrins on the one hand, and tumor formation on the other hand, it should be possible to modify
morphology and behavior of these malignant cells by altering cell-ECM interactions.
Here we show that modification of cell surface p1- and p4-integrins in a 3-dimensional (3-D)
basement membrane (BM) assay (Petersen et al., 1992), influences mammary tissue
morphogenesis and also regulates cell growth and signal transduction. Furthermore, cellular
integrins, when normalized, promote the assembly of adherens junctions and influence the
cytostructure of these cells, thereby implying that these two adhesion systems may be
interconnected. Finally, our results suggest that growth as well as malignant behavior is
regulated at the level of the tissue (acini) organization, i.e., the tissue structure appears to
determine the phenotype which in turn overrides the cellular genotype.
Materials and Methods
Substrates and Antibodies
Commercially prepared EHS matrix (Matrigel, Collaborative Research, Waltham, MA) was used
for reconstituted basement membrane assays, and Vitrogen (rat tail collagen 1), -3 mg/ml
(Vitrogen 100, Celtrix Laboratories), was used for thinly coating the surfaces of culture dishes.
Antibodies used for biochemical analysis and immunostaining studies were as follows: for
immunostaining, immunoblotting, and immunoprecipitation of E-cadherin, a-catenin, and p-
catenin, we used clones 36, 29, and 14, respectively (Transduction Laboratories, Lexington,
NY); for immunostaining of type IV collagen we used clone PHM-12 (Biogenex, San Ramon,
CA); for immunostaining of 01- and a6-integrins we used clones AIIB2 and J1B5 (C. Damsky);
for immunostaining of a3-integrin we used clone P1B5; for immunostaining and
immunoprecipitation of 04-integrin we used clone 3E1; for immunoblot analysis of R 1-integrin
we used clone DF5; for immunoblot analysis of 04-integrin we used polyclonal rabbit serum; for
immunoprecipitation of 0 1-integrin we used polyclonal rabbit serum (all from Chemicon
International, Temecula, CA); for immunostaining of Ki-67 we used clone MIB; for immunoblot
analysis of cyclin D-1 we used clone 17A6-4; and for immunoblot analysis of p21ilPwaf- we used
clone EA10 (all from Oncogene Science, Uniondale, NY). Fluorescence and alkaline
phosphatase-conjugated, unlabeled, and nonspecific rat and mouse IgG's were from Jackson
Laboratories (West Grove, PA) and HRP-conjugated secondaries were from DAKO (Carpinteria,
CA). Antibodies used for integrin function-altering studies within the 3-D reconstituted basement
membrane assay were as follows: for p1-integrin functioninhibition we used clone AIIB2 (C.
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Weaver, V. M.; Petersen, O. W.; Wang, F.; Larabell, C. A.; Briand, P.; Damsky, C. et al. Reversion of the Malignant Phenotype of Human Breast Cells inThree-Dimensional Culture and In Vivo by Integrin BlockingAntibodies, article, April 7, 1997; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc879936/m1/4/: accessed April 19, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.