Edgar C. Henshaw

Specific immunotherapy of advanced renal carcinoma: Evidence for the polyclonality of metastases

Autologous, irradiated (10,000 rads) tumor cells mixed with C. parvumwere given weekly to 14 patients with metastatic renal carcinoma. The tumor tissue had been cryopreserved with DMSO. No significant toxicity was produced. Four patients underwent objective responses, and a fifth had prolonged stabilization (27+ months). Varying responses occurred simultaneously in different metastatic lesions within the same patient. Responding patients usually had an excellent ambulatory status and received greater than 20 × 107tumor cells.

Serum growth factors cause rapid stimulation of protein synthesis and dephosphorylation of eIF-2 in serum deprived Ehrlich cells

In Ehrlich ascites tumor cells maintained in serum-free medium for 16 h the rate of protein synthesis was about 50% of the rate in control (well-fed) cells. The addition of 10% calf serum led to a 1.5- to 2-fold stimulation of protein synthesis within 10 min. Stimulation was effected through a non-transcriptional mechanism which operated at the level of polypeptide chain initiation. The effect was due to non-dialyzable serum growth factors which were sensitive to treatment with dithiothreitol and iodoacetamide. Replacing the 16-h-conditioned serum-free medium with fresh serum-free medium stimulated protein synthesis about 30% in serum-deprived cells, and the effect of these low molecular weight nutrients was additive with the effect of serum factors. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) inhibits protein synthesis by competitively inhibiting the guanine nucleotide exchange factor (GEF), and modulation of the extent of phosphorylation of eIF-2 alpha has been suggested as a probable regulatory mechanism in serum-deprived mammalian cells. We measured the ratio of phosphorylated to total eIF-2 alpha in serum-deprived cells. The ratio was elevated in serum-deprived cells compared to control (serum-fed) cells. eIF-2 was rapidly dephosphorylated in response to serum refeeding and returned to near control levels after 10 min. The rapidity of this response and the close temporal correlation between eIF-2 dephosphorylation and increased rate of protein synthesis provide evidence that eIF-2 plays an important role in the regulation of protein synthesis by serum growth factors.

Phosphorylation of ribosomal protein S6 in the Ehrlich ascites tumor cell: Lack of effect of phosphorylation upon ribosomal function in vitro

The incorporation of 32Pi into the ribosomal protein S6, which is a component of the 40 S ribosomal subunit, was measured in Ehrlich ascites tumor cells growing in suspension culture. During a 2-h incubation period, incorporation was 10-fold greater into S6 of cells growing exponentially in complete medium than in cells in which protein synthesis and growth were inhibited by omission of glutamine from the medium. Since the labeling of other phosphoproteins was not similarly depressed in glutamine-deprived cells, the decreased labeling most likely reflects a specific decrease in phosphate incorporation into S6 rather than a lower phosphate pool specific activity. The cycling of ribosomes is inhibited in glutamine-deprived cells, with an accumulation of monomeric ribosomes indicating an inhibition of protein synthesis initiation. To assess whether the decreased phosphorylation of S6 in the ribosomes of glutamine-deprived cells was responsible for their decreased ability to initiate protein synthesis and enter polyribosomes, a mixture of 14C-labeled ribosomes from rapidly growing cells and 3H-labeled ribosomes from glutamine-deprived cells was added to a rabbit reticulocyte lysate cell-free protein-synthesizing system in which ribosomes cycle actively. The 14C3H ratio was determined in polyribosomes as a measure of the relative ability of the two kinds of ribosomes to initiate protein synthesis. The 14C3H ratio in polyribosomes was found to be identical to the input of labeled ribosomes, indicating an equal ability of ribosomes from growing cells and glutamine-deprived cells to function in initiation. Control studies indicated lack of significant dephosphorylation of S6 during purification of ribosomes and during incubation in the reticulocyte lysate. Thus, a functional difference between more and less phosphorylated ribosomes could not be demonstrated in this system.

TPA stimulates S6 phosphorylation but not protein synthesis in Ehrlich cells.

Increased phosphorylation of ribosomal protein S6 has been extensively correlated with an increased rate of protein synthesis. We report here that under two separate conditions in Ehrlich cells an increase in the level of S6 phosphorylation does not result in any increase in the rate of protein synthesis. 1) In glutamine-deprived cells TPA stimulates S6 phosphorylation but has no effect on the rate of protein synthesis, 2) In cells deprived of serum growth factors, addition of serum stimulates both S6 phosphorylation and protein synthesis while TPA stimulates only S6 phosphorylation. These results show that increased phosphorylation of S6 is not sufficient to cause increased rates of protein synthesis, and suggest that additional factors may play a more direct role.

Active specific immunotherapy with tumor cells and Corynebacterium parvum: a phase I study.

Autologous, irradiated (10,000 rads) tumor cells mixed with C. parvum were given as weekly intracutaneous injections to fifteen patients with residual malignant disease. The toxicity was minimal and distinctly less than has been seen with tumor cell‐BCG immunotherapy. A goal of 4 injections of 107 cells each was possible in only 4 patients because of limitations in methods of disaggregation and quantity of tumor available. The feasibility aspects are discussed and a case report of a prolonged regression is presented.

Somatic cell hybridization of human tumor samples.

Human intraspecific hybrids were formed between tumor cells isolated from both primary and metastatic tumors and a tissue culture adapted cell line, D98OR, a HeLa derivative which is thioguanine and ouabain resistant. Five different tumor types in all were attempted: renal cell carcinoma, colon adenocarcinoma, melanoma, chrondrosarcoma, and hepatocarcinoma. The tumor tissue was either (1) immediately dissociated and fused, or (2) frozen and later thawed, dissociated, and fused. Two different PEG concentrations were used. The results reported here demonstrate that: (1) hybrid tumor cell lines can be made from several types of cancer, (2) unfrozen tumor tissue fused with D98OR by exposure to 50% PEG appears optimal, (3) chromosome loss, as determined by flow cytometry studies of hybrid DNA content, is minimal, and (4) hybrids have characteristics consistent with derivation from tumor cells rather than derivation from the nonmalignant cells of a tumor.

MODULATION OF PROTEIN SYNTHESIS AND EUKARYOTIC INITIATION FACTOR 2 (eIF-2) FUNCTION DURING NUTRIENT DEPRIVAL IN THE EHRLICH ASCITES TUMOR CELL

ABSTRACT We have shown previously that initiation is inhibited in Ehrlich cells deprived of an essential amino acid or of glucose, and that an inhibited step in initiation is the binding of Met-tRNA f to the native 40s ribosomal subunit. This step is mediated by eukaryotic initiation factor 2 (eIF-2). In the present work we have studied the amount and degree of phosphorylation of eIF-2 in the ribosome-bound (KCl wash) and soluble (S-100) fractions of fed, glucose-deprived, and amino acid-deprived cells. The amount of eIF-2, measured by the Millipore filter binding assay, is similar in fed and deprived cells, in both the KCl wash and the S-100 fractions. In cells exposed to 32 Pi for two hours, the 38,000 dalton subunit of eIF-2 is labeled in eIF-2 purified from total ribosomes or from native 40s subunits, but not in eIF-2 purified from the soluble fraction. Function of eIF-2 requires cycling from the free to the ribosome-bound state, and these data suggest that phosphorylation of eIF-2 may play a role in its cycling. The degree of labeling of the 38,000 dalton subunit is similar in eIF-2 of fed compared to nutrient-deprived cells, whether extracted from the total ribosomal KCl wash fraction or from the native 40s subunits. The 48,000 dalton subunit is also phosphorylated in vivo and differences were not seen between fed and deprived cells. If the degree of labeling reflects the extent of phosphorylation of the proteins, these data indicate that phosphorylation of eIF-2 is not the mechanism regulating initiation in glucose- or amino acid-deprived Ehrlich cells. A ribosomal protein of molecular weight 36,000, presumably S6, is much more highly labeled by 32 Pi in ribosomes of fed than of deprived cells. Monomeric ribosomes and polyribosomes are equally labeled.