Faculty Bibliography

Cultured NIH-3T3 cells were transfected with cDNA constructs encoding human epidermal growth factor-receptor (EGF-R)* and two deletion mutants in the extracellular portion of the receptor molecule. One mutant is devoid of 124 amino-terminal amino acids, and the other lacks 76 residues. Mutant receptors were not delivered to the cell surface unless the transfected cells contained also endogenous EGF-Rs, suggesting that receptor interaction complements the mutation and allows surface display of mutant receptors. Immunoprecipitation experiments revealed an association between mutant and endogenous EGF-Rs when both proteins were expressed in the same cell. Hence, receptor-oligomers may exist in the plane of the membrane even in the absence of ligand binding, and oligomerization may play a role in normal trafficking of EGF-Rs to the cell surface. Mutant receptors retained partial ligand binding activity as 125I-labeled EGF was covalently cross-linked to both mutant receptors, and EGF stimulated, albeit weakly, their protein tyrosine kinase activity. Both mutant EGF-Rs bind EGF with a 10-fold lower affinity than that of the solubilized wild type EGF-R. These results provide further evidence that the region flanked by the two cysteine-rich domains plays a crucial role in defining ligand-binding specificity of EGF-R.

In vitro site-directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF)-receptor (i.e. Tyr1173, Tyr1148 and Tyr1068) by phenylalanine, a residue which cannot serve as a phosphate acceptor site. In another mutant, Tyr1173 was substituted by a serine residue. The cDNA constructs encoding either mutant or wild-type EGF-receptors were transfected into NIH-3T3 cells devoid of endogenous EGF-receptors. The mutant receptors were expressed on the cell surface and displayed typical high- and low-affinity binding sites for [125I]EGF. Phorbol ester (PMA) modulated the binding affinity of wild-type and mutant receptors in a similar manner. Mutant EGF-receptors exhibited EGF-dependent tyrosine kinase activity leading to self-phosphorylation and phosphorylation of exogenous substrates both in vitro and in living cells. The internalization and degradation of EGF-receptors were not affected by the mutations. Cells expressing mutant EGF-receptors became mitogenically responsive to EGF, indicating that none of the vital functions of the EGF-receptor were critically impaired by the loss of individual autophosphorylation sites. Maximal mitogenic stimulation correlated with the number of wild-type or mutant receptors per cell, highly expressing cells showing higher maximal stimulation. However, the dose-response curves of cells expressing mutant receptors were slightly shifted to lower concentrations of EGF, rendering the cells mitogenically responsive to lower doses of EGF than cells expressing normal EGF-receptor at similar expression levels. Basal [3H]thymidine incorporation in the presence of 0.5% calf serum was consistently higher for cells expressing mutant receptors, while the response to stimulation with 10% calf serum was not affected.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.

Cultured NIH 3T3 cells devoid of endogenous EGF receptors were transfected with cDNA constructs encoding either the human EGF receptor or an EGF receptor mutant in which Lys721, a key residue in the ATP binding site, was replaced with an alanine residue. The mutant receptor was properly processed, and it displayed both high- and low-affinity surface binding sites. Unlike the wild-type receptor, the mutant receptor did not possess intrinsic protein-tyrosine kinase activity. The initial rate of EGF internalization was similar for wild-type and mutant EGF receptors. Surprisingly, the mutant receptors were not down regulated, but appeared to recycle in transfected cells. These data suggest that degradation of normal EGF receptors after endocytosis is due to the kinase activity endogenous to this receptor. A single amino acid substitution rendered a "down-regulated" receptor into a receptor that can recycle from cytoplasmic compartment back to the cell surface.

Forty-four patients with non-small cell carcinoma of the lung were treated every 3 weeks with vinblastine (4 mg/m2/day iv X 2) and cisplatin (20 mg/m2/day iv X 3). Of the 28 patients with metastatic disease, eight (29%; 90% confidence interval of true response, 17%-47%) achieved objective response, for a median duration of 27 weeks. Median survival in this group was 47 and 28 weeks for responders and nonresponders, respectively. Of the 16 patients with advanced regional disease, 11 (69%; 90% confidence interval of true response, 49%-86%) achieved objective response. Thirteen of these patients received consolidation radiotherapy (4500 cGy/25 fractions/5 weeks), with a boost of 1000 cGy/5 fractions/1 week in those patients who achieved response. In the three patients who did not receive radiotherapy, two died during the induction phase, one from grade 4 leukopenia and sepsis and the second from unrelated factors. The third patient had systemic progression of disease during induction chemotherapy. Six patients experienced overall improvement in their chemotherapy response from the radiotherapy. Two patients who did not respond to the chemotherapy achieved partial response with irradiation. Four patients who had partial response to the chemotherapy achieved complete response with irradiation, and seven patients had no further change in their degree of response to irradiation. The overall median survival of this group was 81 weeks. Maintenance chemotherapy was not given. After radiotherapy, the site of first failure was outside the radiation field in nine of 13 patients (69%). Hematologic toxicity was dose-limiting. Other toxic effects that were not dose-limiting included nephrotoxicity, neurotoxicity, and acute nausea and vomiting. In the patients with advanced regional disease, there was no increase in the radiation toxicity attributable to the chemotherapy. We conclude that: (a) this dose schedule of vinblastine and cisplatin has reproducible activity in non-small cell carcinoma of the lung; (b) the response and median survival of patients with advanced regional disease are superior to those of patients with metastatic disease; and (c) in patients with advanced regional disease, treatment with chemotherapy followed by radiotherapy yielded an overall response rate of 81% (90% confidence interval of true response, 60%-93%) and improved survival compared to a similar group of patients studied by others receiving radiotherapy alone. We recommend further testing of this concept

We hypothesized that cyclophosphamide and cis-platinum, without adriamycin, which had been used in previous studies, may be equally efficacious, but less toxic. We treated 27 patients with non-small cell bronchogenic carcinoma with the combination of cyclophosphamide and cis-platinum. We report six responses (25% response rate), with median survival of 79 weeks as compared to 28 weeks in nonresponders (p less than 0.01). Our regimen had acceptable hematologic toxicity and tolerable gastrointestinal toxicity. However, cumulative nephrotoxicity and neurotoxicity were observed. We conclude that cyclophosphamide and cis-platinum may compare favorably to the cyclophosphamide, adriamycin and cis-platinum combination, with respect to response and toxicity