Expression of epidermal growth factor receptors on human cervical, ovarian, and vulval carcinomas.

We describe the properties of two monoclonal antibodies produced to a synthetic peptide consisting of residues 985 to 996 from the cytoplasmic domain of the epidermal growth factor (EGF) receptor. We have examined a group of ten human tumors including cervical, ovarian, and vulval carcinomas for expression of EGF receptors by immunohistological staining using one of these antibodies and another monoclonal antibody to the extracellular domain of the molecule. The tumors were examined using a sensitive amplified enzyme system and a less sensitive indirect staining method. There was generally a good correlation in staining intensity with the two monoclonal antibody reagents. Both antibodies showed strong staining of squamous cell carcinomas and usually weak or heterogeneous patterns with the adenocarcinomas. Samples of each tumor were solubilized in detergent and analyzed for the presence of functional EGF receptors by immunoprecipitation and autophosphorylation. Three of the squamous cell tumors gave labeled bands, Mr 170,000, on sodium dodecyl sulfate:polyacrylamide gels. DNA was extracted from seven of the tumors and digested with two restriction endonucleases, and the fragments were analyzed on Southern blots using probes representing the extracellular and cytoplasmic domains of the molecule. The tumor DNA showed no apparent rearrangements or amplifications when compared to the EGF receptor gene in human placental DNA. These results suggest that there is a high level of EGF receptors on some squamous cell tumors.

Irregular radiation response of a chondrosarcoma.

The DC II mouse chondrosarcoma is a potentially valuable radiobiological tumour system since it has been observed to recover from radiation injury by regrowth from clones that may be counted in histological sections. Unfortunately, the normal growth of this tumour following s.c. implantation in the thigh is irregular both in the time before growth becomes evident and in the rate of growth. The response to radiation is also unreliable since tumours irradiated with the same dose (e.g. 30 Gy) show a range of responses from shrinkage to no detectable change in growth rate. The delay in normal growth can be attributed largely to delays in vascularization while changes in growth rate may be explained by differences in tumour architecture. Radiation response may depend on variations in hypoxic fraction and in relative cellularity. Tumours having the same external dimensions may differ by a factor of 80 in the numbers of tumour cells they contain. This chondrosarcoma may prove a closer model to some human tumours than many transplantable tumours that display regular growth patterns.

A radioimmunoassay for human epidermal growth factor receptor.

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.

The use of anti-synthetic peptide antibodies to study the v-erb B protein from chicken and rat cells transformed by avian erythroblastosis virus.

Two site-specific anti-peptide antisera have been produced that efficiently recognize the native form of the v-erb B protein from avian erythroblastosis virus (AEV)-infected chicken erythroblasts and fibroblasts, and from AEV-transformed mammalian cells. Since the antibodies were generated against synthetic sequences, the immunoprecipitations could be performed in the presence or absence of immunizing peptide, permitting the specifically precipitated proteins to be identified from background non-specifically adsorbed proteins. We confirmed that immobilized v-erb B protein from cell lysates of unlabelled AEV-infected chicken erythroblasts became labelled upon incubation with [gamma-32P]ATP. In addition we demonstrated for the first time that v-erb B from mammalian cells became labelled under the same conditions. These results suggest that the v-erb B protein may possess intrinsic kinase activity. The reagents described should permit further investigations as to whether this activity plays a role in maintaining cellular transformation.

Isolation and nucleotide sequence of a cloned cardionatrin cDNA.

A cloned cDNA which codes for the C-terminal 62 residues of the precursor molecule for the atrial natriuretic factor, cardionatrin, has been isolated and sequenced. The nucleotide sequence confirms the amino acid sequence of cardionatrin 1-28 previously determined, and positions this peptide at the C-terminal end of the precursor just two residues away from the termination codon.