Unconjugated
A comparative study of six mouse monoclonal antibodies against human 45 kDa keratin polypeptide (keratin No. 18) was undertaken using three experimental approaches: immunohistochemistry on normal human tissues, examination of interspecies cross-reactivity and identification of the target polypeptides in 1-D and 2-D immunoblots. The data suggest that at least five different antigenic sites of keratin 18 are recognized by this panel of reagents. The C-04 epitope is keratin 18-specific and widely conserved among mammalian species, while the antibodies DA7 and DC10 also react specifically with the 45 kDa keratin but stain simple epithelia of human origin only. Two antibodies, C-11 and C-66, decorate simple as well as stratified epithelia in human and in all seven animal species tested, but their respective target epitopes are shared by different groups of keratin polypeptides, which indicates their non-identity. In contrast to keratin specificity of the five above mentioned antibodies, the C-08 antibody cross-reacts with a 70 kDa nuclear lamina protein found in human and bovine tissues. The results of the present study provide the necessary basis for future applications of these antibodies in both routine immunodiagnostic work and as probes to study the biology of epithelial cells in general and the significance of keratin intermediate filaments in particular.
The luminal and basal epithelial cells in the human mammary gland can be distinguished in tissue sections on the basis of the pattern of keratins they express. Moreover, the invasive cells in primary carcinomas show a keratin profile that corresponds to that of the dominant luminal cell (7, 8, 18, 19). When homogeneous populations of luminal epithelial cells from milk or from breast cancer metastases are cultured the profile of keratin expression seen in vivo is maintained. We have therefore used monospecific antibodies reactive with individual keratins to examine the phenotype of cells cultured in three different media from reduction mammoplasty tissue that contains both luminal and basal cells. The phenotype of cells cultured from primary breast cancers in one of these media (MCDB170) has also been examined. In characterizing cell phenotypes, antibodies to a polymorphic epithelial mucin (PEM) expressed in vivo by luminal cells, and to smooth muscle (a) actin, expressed in vivo by basal cells, have also been used. Our results show that proliferation of different cell phenotypes is selected for in different media. In milk mix (MX) developed for growth of luminal cells from milk, only the luminal cell phenotype proliferates (for only 1 or 2 passages). In medium MCDB 170, which was developed for long-term growth of human mammary epithelial cells from reduction mammoplasty organoids, cells from the basal layer proliferate, while in MM medium the basal phenotype dominates, but a few cells with the luminal phenotype are found. Around passage 3, in medium MCDB 170, most cells senesce and a subpopulation of cells proliferates on further passage. These cells retain expression of the basal epithelial keratins but also express some features characteristic of luminal epithelial cells, suggesting that the basal layer may contain a stem cell that can develop along the luminal lineage. In culture, however, they do not express keratin 19, which in vivo is a feature of the fully differentiated luminal cell. The cells cultured from primary breast cancer in medium MCDB 170 have a similar keratin profile to that of the normal cells cultured in this medium. They do not express keratin 19, even though the invasive cells in primary cancers homogeneously express this keratin in vivo. The invasive phenotype, which in its keratin profile corresponds to the differentiated luminal cell and that of the metastatic cancer lines, cannot be cultured from primary breast cancers using MX, which supports proliferation of the corresponding normal cell.