Negative cooperativity between alpha 3 beta 1 and alpha 2 beta 1 integrins in human mammary carcinoma MDA MB 231 cells.

The alpha 3 beta 1 integrin has been implicated as a receptor for several matrix components, including collagen, fibronectin, and laminins. The function of alpha 3 beta 1 seems to be very versatile involving cell adhesion to or migration on ECM, establishment of cell-cell contacts in aggregates, as well as linkage to intracellular tyrosine phosphorylation cascades. Here we report a strong induction of attachment of alpha 3 beta 1 integrin expressing human breast carcinoma cell line MDA MB 231 to matrix proteins by two alpha 3 integrin subunit function-blocking monoclonal antibodies (P1B5 and ASC-1). In contrast, stimulation of adhesion to ECM by inhibitory alpha 3 integrin-specific antibodies was not observed in the alpha 3 beta 1 integrin-expressing nonmalignant human mammary epithelial cell line MCF-10A or the human breast carcinoma cell line MDA MB 468 that expressed relatively low amounts of alpha 3 beta 1 integrin at the cell surface. This increase was specific for collagens and not observed on fibronectin or laminin. Physiological concentrations of bivalent cations were not required. MAb P1B5 did not induce homotypic aggregation of MDA MB 231 cells. The P1B5-induced increase in cell attachment to collagens could be prevented but not reduced below control levels by blocking mAb to the alpha 2 integrin subunit. Function blocking anti-alpha 5 integrin subunit mAb was without effect while anti-beta 1-mAb completely abolished adhesion. Our data indicate that negative cooperativity between integrins results in transdominant inhibition of alpha 2 beta 1 function by alpha 3 beta 1 in human MDA MB 231 but not MDA MB 468 tumor cells or nonmalignant MCF-10A cells.

Construction of a chimeric antibody with therapeutic potential for cancers which overexpress c-erbB-2.

We describe the chimerization of a monoclonal antibody directed against the c-erbB-2 protein using a novel PCR method for cloning immunoglobulin variable region genes. We also describe the characterization of the chimera and show its potential use for treating cancers which overexpress the c-erbB-2 protein. The genomic DNA fragments of heavy and light chain variable genes were cloned by PCR using uniquely designed primers which allowed for isolation of genes containing functional promoters, signal and coding sequences. The chimeric genes were then constructed by linking variable regions of murine genes to human constant gamma 1 and kappa genes. Expression of the chimeric immunoglobulin genes resulted in production of properly assembled chimeric antibody with improved biological properties.

Development of resistance to cisplatin is associated with decreased expression of the gp185c-erbB-2 protein and alterations in growth properties and responses to therapy in an ovarian tumor cell line.

Overexpression of the c-erbB-2 protein (gp185c-erbB-2) is correlated with a tumorigenic phenotype and may contribute to disease progression. We have reported previously on an anti-gp185c-erbB-2 antibody, TAb 250, that inhibits in vitro and in vivo growth of breast and ovarian cell lines that overexpress the protein and enhances the inhibitory activity of cisplatin (CDDP). To assess whether CDDP resistance is related to gp185c-erbB-2 expression levels, alterations in tumor cell growth characteristics, or efficacy of antibody plus drug combination treatments, an SKOV-3 ovarian tumor cell line was made resistant to escalating doses of CDDP. Parental cells were 12-fold more sensitive to CDDP with 7 times more gp185c-erbB-2 sites than the most resistant variant (SKOV-3/C12). Additionally, the resistant cells demonstrated a longer lag phase for in vivo growth than the parental cells. While TAb 250 enhanced the in vivo inhibitory effect of CDDP against parental SKOV-3 cells, the antibody did not significantly alter the CDDP responsiveness of the resistant population. Growth inhibition by TAb 250 alone of both the parental and the SKOV-3-resistant variants was similar; however, TAb 250 was able to prolong the lag-phase of tumor growth of the resistant variant by up to 25 days. These results indicate that the development of CDDP resistance is associated with lowered levels of gp185c-erbB-2 expression, slower tumor cell growth, and enhanced efficacy of antibody treatment of the resistant cells.

Determination of renal molar concentrations of phosphorus-containing metabolites in vivo using 31P NMR.

A technique to determine absolute metabolite concentrations of the kidney in vivo using 31P NMR is described. The technique is based on the use of methylphosphonic acid (MPA), which gives rise to a well-resolved peak upfield from in vivo phosphorous metabolite resonances, as an "internal standard." The method involves acquisition of a fully relaxed kidney spectrum with an implanted coil followed by intravenous infusion of MPA (4 ml of 150 mM) for a period of 30 min. The animal is then sacrificed to insure a steady state level of renal MPA and another spectrum is obtained. From these two spectra the ratio of intensities of MPA to beta-ATP was derived. In the method used here, no significant contribution from tissues outside the kidney was observed. In addition, a relatively homogeneous distribution of MPA throughout the kidney was achieved. The amount of MPA per gram wet weight of kidney was also obtained through NMR methods by placing the excised organ in a phosphate-calibrated solenoidal coil. The calibration spectra along with the ratio of intensities for MPA/beta-ATP were used to calculate the number of micromoles of ATP per gram wet weight of kidney. Infusion of a higher concentration of MPA (1.25 M) produced a visible MPA resonance in other organs besides the kidney. Thus, MPA could be useful in determining phosphate metabolite concentrations in other tissues.