A major, apparently novel extracellular matrix-degrading protease was previously identified and partially isolated from hormone-dependent but not from hormone-independent human breast cancer cells (Shi, Y. E., Torri, J., Yieh, L., Wellstein, A., Lippman, M. E., and Dickson, R. B. (1993) Cancer Res. 53, 1409 -1415). Although initially the 80-kDa protease was identified from breast cancer cell-conditioned medium, immunofluorescence staining of breast cancer cells with anti-80-kDa protease monoclonal antibody 21-9 showed that in addition to its detection in intracellular compartments, the protease was uniformly localized around periphery of the cells with more intensive staining on the pseudopodia and membrane ruffles. A surface biotinylation technique confirmed the plasma membrane localization of the protease. In addition, the 80-kDa protease could not be washed from the membrane fraction of homogenized breast cancer cells with high concentrations of salts or with EDTA.The 80-kDa protease may noncovalently associate with other protein(s) to form complexes, the 95-and 110-kDa proteases. Both complexes showed gelatinolytic activity and bore the epitopes recognized by monoclonal antibody 21-9. Furthermore, both complexes could be converted to 80-kDa forms by boiling in SDS in the absence of reducing agents. Expression of this novel, integral membrane gelatinase could allow breast cancer cells an alternative to other previously described matrix-degrading enzymes for degradation of the extracellular matrix in close proximity to their surfaces.Fatality in breast cancer is the result of metastatic lesions. Degradation of the extracellular matrix (ECM), 1 including the basement membrane and interstitial stroma, an important aspect of metastasis, is required for metastatic cancer cells to migrate through anatomical barriers and to invade tissues. Matrix-degrading proteases, including the plasminogen activator-plasmin system of serine proteases, 72-kDa gelatinase A (MMP-2) and 92-kDa gelatinase B (MMP-9) of the matrix metalloprotease (MMP) family, and cathepsins B (a cysteine protease) and D (an aspartic protease), have been implicated in tumor cell invasion (2). Morphological studies show that degradation of the ECM occurs in close proximity to the cell surface (3, 4), and that matrix-degrading proteases must be localized on the cell surface to perform their functions. This theory was further supported by the observation that the cancer cellconditioned medium alone failed to degrade the ECM (5). Studies of two matrix-degrading proteases, the plasminogen activator-plasmin system and the activation of 72-kDa gelatinase A (MMP-2), clearly demonstrate that localized ECM degradation is achieved by protease binding to a membrane-bound protease receptor and/or activator followed by a proteolytic activation cascade. The urokinase-type plasminogen activator (uPA) is secreted as a soluble, inactive precursor by tumor cells and/or tumor stromal cells and recruited to tumor cell surfaces by binding to the membrane-bound uPA receptor. ...

Marimastat was well absorbed from the gastrointestinal tract, with high levels of the study drug detected in plasma within hours after drug administration. Plasma concentrations of Marimastat achieved at dose levels 2 and 3 (50 mg and 100 mg orally twice daily) were substantially higher than those required for MMP inhibition in vitro. The dose-limiting toxicity (DLT) was severe inflammatory polyarthritis, which seemed to be a cumulative toxicity.

We have examined defects in mammary development and tumorigenesis in a transgenic model expressing the c-myc gene under the MMTV ± LTR promoter. The stochastic tumors which arise from hyperplastic ductal and lobular lesions in this model are characterized by high rates both of apoptosis and of chromosomal instability. Since the p53 gene product is thought to be central in the maintenance of genomic integrity, in part due to its ability to induce apoptosis in cells harboring DNA damage, we examined its expression and possible mutation. Initially, we observed that unmutated p53 is strongly expressed in premalignant mammary glands and in mammary tumors derived from the MMTV-c-myc strain. We then mated the MMTV-myc strain to a p53-de®cient strain as a means of examining the eect of this lesion on mammary development and tumorigenesis in the context of c-myc overexpression. A lack of both p53 alleles in the presence of c-myc overexpression resulted in a dramatic hyerplastic alteration in mammary gland development. Speci®cally, in female bitransgenic MMTV-c-myc/p53 null mice (MMTV-myc/p53 7/7 ), lobular hyperplasias were observed at almost every ductal end bud as early as 32 days of age. In contrast, only mild ductal and lobular hyperplasias were seen in MMTV-myc mice that contained both p53 alleles (MMTV-myc/p53 +/+ ); an intermediate phenotype occurred in mice with a single intact (MMTV-myc/p53 +/7 ) p53 allele. Mammary carcinomas arose with a high frequency in MMTV-myc/p53 +/7 mice; the tumors were comparable in frequency, histology and apoptotic index to the tumors in MMTV-myc/p53 +/+ mice. Also, as previously observed (Elson et al., 1995), lymphomas arose with extremely short latency in MMTV-myc/ p53 7/7 mice, precluding study of the fate of their hyperplastic mammary lesions in situ. The frequency of p53 mutations in MMTV-myc/p53 +/+ and MMTV-myc/ p53 +/7 mammary tumors and in cell lines derived from these tumors was examined by direct sequencing. No point mutations or deletions in p53 were observed in mammary tumors or cell lines from either genotype. Finally, a detailed chromosomal analysis using multicolor spectral karyotyping (SKY) revealed that there were multiple chromosomal alterations in the c-mycoverexpressing cells that contained either one or two unmutated p53 alleles. Variable ploidy changes, a common translocation of chromosome 11, and other chromosomal aberrations were observed. Our data thus support an interaction between c-Myc and p53 in mammary development, but suggest that loss of p53 is required neither for c-myc-dependent tumorigenesis nor for c-myc-dependent chromosomal instability.