Detection of nuclear matrix proteins in serum from cancer patients.

Morphological characteristics of the cell nucleus have long been used by pathologists in the clinical diagnosis of cancer. The nuclear matrix of the cell, the structure that serves to organize the chromatin within the nucleus, is known to reflect these morphological characteristics with regard to cell and cancer type. Monoclonal antibodies were developed to extracted nuclear matrix proteins. These antibodies were used in two-site immunometric assays to detect soluble nuclear matrix proteins in the supernatants of two dying cell lines and 15 tumor tissues. Furthermore, nuclear matrix proteins were detected at elevated levels in the sera of cancer patients compared with normal patient sera. In one assay, 63.2% of cancer sera read above 95% of normal sera, and in another assay, 73.7% of cancer sera read above 95% of the normal sera. With the development of monoclonal antibodies with greater cancer specificity, the detection of circulating nuclear matrix proteins may become an important clinical tool in the diagnosis and monitoring of cancer.

Expression of the TGF alpha integral membrane precursor induces transformation of NRK cells.

TGF alpha is one member of a family of soluble growth factors that are derived from integral-membrane precursors. The mature form of TGF alpha is released from its transmembrane precursor (proTGF alpha) by a protease that, in many tumor cells, is inefficient or limiting. We have previously established that, in the absence of processing, membrane-anchored proTGF alpha is biologically active and can interact with the EGF receptor on adjacent cells, thereby inducing the receptor's intrinsic tyrosine kinase activity. We further showed that this interaction leads to immediate downstream signal transduction as evidenced by Ca2+ mobilization. To extend these observations, and to investigate its transforming potential, we infected normal rat kidney (NRK) cells with retroviral expression vectors that encode mutated forms of proTGF alpha containing amino acid substitutions at the proteolytic cleavage sites. NRK cells harboring these mutant constructs do not secrete mature growth factor, but do express biologically active proTGF alpha on the cell surface as shown by their ability to induce the autophosphorylation of EGF receptor on neighboring A431 cells in co-culture. Expression of the mutant proTGF alpha molecules promoted the anchorage-independent growth of NRK cells in soft agar, and caused them to be tumorigenic when injected into nude mice. These results demonstrate that an interaction between EGF receptor and the integral membrane precursor to TGF alpha can provide a mitogenic stimulus that leads to transformation. They further suggest that the accumulation of proTGF alpha on the surface of some transformed cells has physiological relevance.

Differential induction by interferons of major histocompatibility complex-encoded and non-major histocompatibility complex-encoded antigens in human breast and ovarian carcinoma cell lines.

Treatment of cancer cells with interferons can modulate expression of cell surface antigens, particularly those of the major histocompatibility complex (MHC). To examine the effect of recombinant gamma- and alpha-interferons on expression of non-MHC antigens, murine monoclonal antibodies have been used to quantitate 14 distinct tumor-associated cell surface antigens from five breast cancer cell lines and five ovarian cancer cell lines using a live cell radioimmunoassay. Both Class I and Class II MHC antigens could be augmented or induced with gamma-interferon. Significantly increased expression of MHC antigens was observed in nine of 10 cell lines with induction indices as high as 11-fold. When 17 non-MHC epitopes were measured on 10 cell lines, minimal (1.3-2.7-fold) induction was observed in 10 of the 170 instances evaluated. Expression of only two epitopes, 2G3 and 735B11, was increased on more than one cell line. On six cell lines expression of non-MHC epitopes could not be increased. Consequently, among many different cell surface determinants, interferons produced a highly selective augmentation or induction of MHC antigens, whereas augmentation or induction of other tumor-associated antigens was apparently restricted to a few epitopes.

The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction.

The 50 amino acid form of TGF-alpha is cleaved from a conserved integral membrane glycoprotein by a protease that, in many tumor cells, appears to be limiting. To test whether the membrane-bound precursor has biological activity in the absence of processing, we introduced amino acid substitutions at the proteolytic cleavage sites. BHK cells transfected with expression vectors containing these altered sequences do not secrete detectable levels of mature TGF-alpha into the medium, but express high levels of proTGF-alpha at the cell surface. Coincubation of these BHK cells with A431 cells demonstrates that membrane-bound proTGF-alpha may bind to EGF receptors on the surface of contiguous cells, induce receptor autophosphorylation, and thereby produce a rapid rise in A431 intracellular calcium levels. Thus, proTGF-alpha can be biologically active in the absence of processing, a fact that may have implications for the integral membrane precursors of related growth factors.