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Monoclonal antibodies with specificity for mucin-like antigens have shown great promise for the diagnosis and therapy of human cancer. Heterogeneity in the expression of mucin-like antigens by tumor and normal cells has been noted in several previous studies. An understanding of the nature of this heterogeneity has important implications for the diagnostic and therapeutic usefulness of antibodies to mucin-like antigens. We have studied the mechanism of variability in expression of epitopes on a mucin-like antigen defined by monoclonal antibodies W1, W5, and W9 in the lung carcinoma cell line, Calu-1. Using the fluorescence activated cell sorter and clonal analysis, we have demonstrated that intercellular variability in mucin antigen expression by Calu-1 cells can be explained in part by heritable variation in the tumor cell population. Clonal cell lines were isolated which differ greatly in levels of epitopes for all three mucin directed antibodies. Levels of all three epitopes showed significant variation between different clonal lines but in general were coordinately regulated. Differences in epitope expression between two lines studied in detail could be attributed to a dramatic difference in expression of a high molecular weight mucin-like glycoprotein. In immunoblotting experiments the binding of all three antibodies to this glycoprotein was affected by sodium periodate and/or neuraminidase treatment, suggesting that the antibodies recognize carbohydrate epitopes. Thus, heterogeneity in expression of mucin-like glycoprotein antigens can result from heritable variations which affect expression of multiple carbohydrate epitopes.

We have detected restriction fragment length polymorphisms associated with the immunoglobulin heavy chain C gamma genes. DNA from both parents of an individual having an unbalanced rearrangement of the long arm of chromosome 14, region q32 [Cox, D. W., Markovic, V. D. & Teshima, I. E. (1982) Nature (London) 297, 428-430], revealed distinctive patterns of BamHI fragments which hybridized with cloned probes from the C gamma 2-C gamma 4 gene cluster. The number of hybridizing fragments in both cases (five) equaled the number of known C gamma genes. Pedigree and densitometric analyses indicated that the proband did not have any maternal complement of C gamma gene-hybridizing fragments. Included on the deleted chromosomal segment was a C gamma gene having properties of the previously reported C gamma pseudogene. We also examined DNA from this family with a probe for the highly polymorphic locus D14S1, which recently was demonstrated to be tightly linked to the C gamma 1 gene locus [Balazs, I., Purrello, M., Rubinstein, P., Alhadeff, B. & Siniscalco, M. (1982) Proc. Natl. Acad. Sci. USA 79, 7395-7399]. EcoRI and EcoRI-BamHI fragments from both parents hybridized with a probe for this locus in DNA from the proband, indicating that, unlike the C gamma gene family, D14S1 was not deleted from the abnormal chromosome. Thus, the chromosomal breakpoint in the proband lies within region 14q32 between the two tightly linked markers, D14S1 and the C gamma 1 heavy chain gene locus. The D14S1 locus must lie proximal to the centromere relative to the C gamma gene family. The genetic variability detected with C gamma gene probes may prove useful for genetic analysis of structural rearrangements involving this region of chromosome 14.

Epidermal growth factor (EGF) initiates a wide variety of events when added to responsive cultured cells. These range from early events requiring only brief exposure to EGF, e.g., stimulation of transport of amino acids or ions, to later events such as commitment of cells to a round of DNA synthesis, a process requiring 6 h or more of continuous exposure to hormone. EGF binding is followed first by phosphorylation of EGF receptors, which can be detected in purified membranes and permeabilized cells, and then by internalization and proteolytic processing of receptors in lysosomes. Native 160,000-dalton EGF receptors contain a site that is not exposed on the cell surface and is highly sensitive to cleavage by an endogenous protease, which yields a 145,000-dalton receptor fragment that retains phosphate acceptor activity. Cleavage of receptor at a trypsin-sensitive site, also not exposed to the cell surface, yields a 115,000-dalton fragment that binds EGF, but contains no phosphorylated species. The data indicate that the phosphate acceptor sites on EGF receptors are localized on a 45,000-dalton cytosolic region.

We have studied the biosynthesis of mucin glycoproteins recognized by monoclonal antibody W1 in MCF7 cells. Proteins of Mr 170,000, 185,000, 260,000, and 275,000 were immunoprecipitated from cells pulse-labeled with [3H]threonine and [3H]proline. Evidence suggesting that these proteins were precursors of high molecular weight mucin(s) included: 1) their kinetics of disappearance corresponded with appearance of mature mucin(s) (t1/2 = 30 min); 2) their processing into mature mucin(s) was not blocked by cycloheximide, but was disrupted by monensin, which impairs glycoprotein processing in the Golgi; 3) they were inaccessible to antibody added outside the cells, whereas mature mucin(s) was accessible and appeared at the cell surface with a t1/2 = 45 min; and 4) their mobilities of precursors varied between different cell lines, but generally correlated with mobilities of mature mucin(s). The precursors were sensitive to endoglycosidase H, indicating that they contained high mannose N-linked oligosaccharides. Less than 3% of threonine residues in the precursors, but more than 75% in mature mucin(s), were substituted with O-linked oligosaccharides. Therefore, initiation of N-linked oligosaccharides occurred soon after initiation of core protein synthesis, but initiation of O-linked oligosaccharides occurred much later, just prior to appearance of mature mucin(s) at the cell surface.