Peter M. Blumberg

Specific binding of phorbol ester tumor promoters to mouse skin

Abstract The tumor promoter 20- 3 H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (K D = 2.4 × 10 −8 M). The potencies of phorbol esters for inhibiting binding of 3 H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, K I = 0.74 nM; phorbol 12,13-didecanoate, K I = 16 nM; phorbol 12,13-dibenzoate, K I = 82 nM; mezerein, K I = 98 nM; phorbol 12,13-diacetate, K I = 3 μM; phorbol 12,13,20-triacetate, K I = 5.6 μM; phorbol 13-acetate, K I = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, 3 H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the 3 H-PDBu binding activity mediates phorbol ester tumor promotion. 3 H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE 2 , 1 μM) some of the effects of the phorbol esters in vivo or in vitro.

Analysis of phorbol ester receptors in phorbol ester unresponsive 3T3 cell variants

Abstract The phorbol ester tumor promoters induce multiple cellular responses in cell culture, including mitogenesis. We have analyzed 3 variants of mouse 3T3 cells mitogenically unresponsive to the phorbol esters for phorbol ester receptors. All resembled control 3T3 cells in their specific [3H]phorbol 12,13-dibutyrate binding. The variants thus appear to be altered at steps distal to receptor occupancy in the mitogenic response to the phorbol esters.

Regional localization ny light microscopic autoradiography of receptors in mouse brain for phorbol ester tumor promoters

The receptors for the phorbol ester tumor promoters are present in highest concentration in the brain. We have examined the distribution of receptors in mouse brain by light microscopic autoradiography. Marked differences in receptor levels were found both between brain regions and between layers of a single region. Binding was high in hippocampus, cerebral cortex, striatum and substantia nigra, lower in thalamus and pons. In cerebellum, binding was high in the stratum moleculare, intermediate in the stratum granulosum and low in the lamina alba. The localization suggests a functional role for the receptors within brain, and it permits comparison with other known activities. For example, the pattern of localization is distinct from those reported for neurotransmitter receptors or for phospholipids.

How Penicillin Kills Bacteria: Progress and Problems

Penicillins and cephalosporins are specific inhibitors of the biosynthesis of bacterial cell walls. This discovery was first made in 1957 and was based on two observations. First, penicillins induced the formation of protoplasts or spheroplasts in bacteria (organisms in which the cell wall has been lost or weakened) (Lederberg 1957). Secondly, a uridine nucleotide accumulated in Staphylococcus aureus and other bacteria inhibited by penicillin which had a striking relationship to the composition of the cell wall (Park & Strominger 1957). It was therefore suggested that this nucleotide was an activated precursor of the wall. Over the next decade, a great deal of work was carried out in order to elucidate the structure of the bacterial cell wall and the mechanism of its biosynthesis from the uridine nucleotides and other precursors (reviewed by Strominger 1970; Strominger & Ghuysen 1967; Ghuysen 1968). It was demonstrated that interpeptide cross-links were an important structural feature of the wall. Several kinds of experiments carried out with whole cells indicated that the final step in cell wall synthesis, the crosslinking reaction catalysed by a transpeptidase, was the site of action of penicillin (Wise & Park 1965; Tipper & Strominger 1965 a, b, 1968). Finally, in 1966, the transpeptidase catalysing this cross-linking reaction was obtained in a cell-free system and shown to be a penicillin-sensitive enzyme (Izaki, Matsuhashi & Strominger 1966, 1968). The history of these developments has been reviewed elsewhere (Strominger 1970), and in the present paper, attention will be focused on recent studies of the penicillin-sensitive transpeptidase and other penicillinsensitive activities found in bacterial cell membranes. First, however, it is necessary to describe briefly the structure of the cell wall of bacteria and the nature of the inhibited reactions. The walls of bacteria consist of glycan strands in which two sugars, acetylglucosamine (X) and acetylmuramic acid (Y), strictly alternate (figure 1). Four such glycan strands are represented in figure 1. The acetylmuramic acid residues of the polymer are substituted by a tetrapeptide (represented in the figure by open circles). The peptidoglycan strand (i.e., the glycan substituted by the tetrapeptide) are cross-linked to one another by means of an interpeptide bridge which is to some extent a genus-specific characteristic. In the genus Staphylococcus aureus, the interpeptide bridge is a pentaglycine chain (represented in figure 1 by the closed circles) which extends from the carboxyl group on the terminal D-alanine residue of the tetrapeptide to the ∊-amino group of lysine, the third amino acid in the tetrapeptide chain. The wall of S. aureus is a very tightly knit structure in that virtually every peptide subunit is cross-linked to another subunit by means of this interpeptide bridge. Penicillins and cephalosporins are specific inhibitors of the reaction in which the cross-link is actually formed. This step is the last reaction in wall synthesis.

Tissue and species specificity for phorbol ester receptors.

Tumor promoters are agents that, although not themselves carcinogenic, induce tumors in animals previously treated with a subthreshold dose of a carcinogen (1–3). Although tumor promotion has been characterized in greatest detail for mouse skin, it has also been demonstrated for the liver, bladder, colon, trachea, and mammary gland (4,5). The potential importance of tumor promotion in human cancer etiology is suggested by a growing body of epidemiological evidence (6–8).

Biological responsiveness to the phorbol esters and specific binding of [3H]phorbol 12,13‐dibutyrate in the nematode Caenorhabditis elegans, a manipulable genetic system

Because of its suitability for genetic studies, the nematode Caenorhabditis elegans was examined for its responsiveness to the phorbol esters. Phorbol 12-myristate 13-acetate had three effects. It inhibited the increase in animal size during growth; it decreased the yield of progeny; and it caused uncoordinated movement of the adult. The effects on nematode size, progeny yield, and movement were quantitated. Concentrations of phorbol 12-myristate 13-acetate yielding half-maximal responses were 440, 460, and 170 nM, respectively. As was expected from the biological responsiveness of the nematodes, specific, saturable binding of phorbol ester to nematode extracts was found. [3H]phorbol 12,13-dibutyrate bound with a dissociation constant of 26.8 +/- 3.9 nM. At saturation, 5.7 +/- 1.4 pmole/mg protein was bound.

Design and construction of a simple and inexpensive slab gel electrophoresis apparatus

Abstract A very simple and inexpensive slab gel electrophoresis apparatus is described. This integral design reduces the leakage, cost, and size limitations frequently encountered in the construction and use of currently available apparatuses. An additional refinement eliminates the need for notching one member of the usual pair of glass plates used as gel slab molds. The apparatuses, in which linear, gradient, and two-dimensional gels have been routinely run, can be built in a wide variety of sizes and shapes for either analytical or preparative purposes. Several gel apparatuses can be clamped together and run simultaneously from a single power source. Ease of construction permits more than a dozen apparatuses of this design to be built in the space of a day or two by unskilled personnel.

PENICILLIN BINDING COMPONENTS OF BACTERIAL CELLS AND THEIR RELATIONSHIP TO THE MECHANISM OF PENICILLIN ACTION

Penicillin is believed to kill bacteria by inhibiting the terminal reaction in cell wall biosynthesis, namely, cross-linking of the cell wall by the membranebound transpeptidase.l The enzymatic mechanism proposed for the transpeptidase reaction is as follows (FIGURE 1 ) . 2 A reactive group on the enzyme, possibly a cysteine, would cleave the peptide bond between the D-alanines in the pentapeptide chain of the uncross-linked cell wall, which liberates the terminal Dalanine and forms an acyl-enzyme intermediate. The amino group from the diaminopimelic acid residue of a nearby peptide chain would then react with the acyl-enzyme intermediate to form a peptide cross-bridge and regenerate free enzyme. In most, but not all, organisms, this reaction is irreversibly inhibited by penicillins. At the same time that the transpeptidase was first assayed in vitro, a second penicillin-sensitive enzymatic activity, that of a D-alanine carboxypeptidase, was also ~ b s e r v e d . ~ ~ This activity resembled that of the transpeptidase in that the terminal D-alanine was released from the pentapeptide chain of the precursor. Cross-linking did not occur in this case, however. Two possible explanations for the presence of the carboxypeptidase activity were advanced. First, the function of the carboxypeptidase might be to liniit the degree of cross-linking of the bacterial cell wall. The product of the carboxypeptidase reaction, the tetrapeptide, would be incapable of serving as a donor in transpeptidation. In accord with this hypothesis, the cell walls of Esrherichici coli and of the Bacilli, organisms that possess carboxypeptidases, are loosely cross-linked. The cell walls of Staphylococcus azireus, which lacks a carboxypeptidase, are highly cross-linked. Second, as suggested by Izaki and colleagues,:; the carboxypeptidase activity that was measured might represent assay of an “uncoupled transpeptidase.” The enzyme would react to release malanine and form an acyl-enzyme intermediate, as with the transpeptidase. In this case, though, water rather than an amino group would then act to displace the enzyme from the acyl-enzyme intermediate. Considerable effort has been devoted over the past 5 years to investigating the relationship between carboxypeptidase and transpeptidase. In Bacillus subtilis, the carboxypeptidase appears to be a distinct protein from the penicillin killing site, which presumably is the transpeptidase.

SUBUNIT STRUCTURE OF SURFACE AND SHED LARGE, EXTERNAL, TRANSFORMATION‐SENSITIVE PROTEIN OF CHICK EMBRYO FIBROBLASTS*

Several laboratories have recently reported that large, external, transformation-sensitive (LETS) glycoprotein exists as disulfide-linked polymers on the sur face of chick embryo fibroblast^^^^ and Nil hamster c e k 3 For the chick fibroblast system, quantitation after lactoperoxidase-catalyzed iodination indicated that 21.3 =t 1.3% of total LETS protein was present as the dimer and 77.4 f 1.9% as the oligomer (mean values + SEM; n = 20) . The term oligomer refers to polymeric LETS protein that was too large to enter a 3.5% sodium dodecyl sulfate (SDS) polyacrylamide gel. These results do not appear to be an artifact of the lactoperoxidase labeling procedure. Alkylation of cells with iodoacetamide either before or after iodination did not affect the proportion of LETS protein dimer; quantitation of the LETS protein dimer by protein staining with Coomassie brilliant blue yielded values similar to those obtained by iodination. Trapping of LETS protein dimer likewisc cannot explain the apparent presence of the oligomer. The quantitation of LETS protein dimer by protein staining was linear with respect to the amount of protein added, thus ruling out concentration-dependent aggregation. When purified radioactive LETS protein dimer was added back to the nonradioactive cell lysate, less than 6% of the radioactivity was retained at the top of the gel under the conditions routinely employed for measuring the proportion of L,ETS protein dimer. The subunit structure of the LETS protein o n the cell surface exhibited little sensitivity to growth conditions. Chick embryo fibroblasts cultured for I , 2 , or 3 days to equal final cell densities showed no difference in the proportions of the LETS protein dimer and oligomer. Growth of cells for 2 days to final cell densities that ranged from 1 to 20 x lo4 cells/cm2 caused the proportions of LETS protein dimer to vary by no morc than 40,;. LETS protein is known to be shed by fibroblasts into the medium. We have examined the subunit structure of this shed material. Chick embryo fibroblasts were labeled by lactoperoxidase-catalyzed iodination, then incubated for 20 hr with conditioned culture medium. Only 23.5 i 3.37; (mean value i SEM; n = 7) of the LETS protein released into the medium was present as the dimeric form. The remainder was primarily oligomer. Comparable results were obtained i f incubation occurred in fresh rather than in conditioned medium. Controls ruled out the possibility of LETS protein dimer trapping due to high serum protein concentration in the medium samples. Preliminary experiments indicate, moreover, that the subunit structure of the shed LETS protein was not altered if the incubation was prolonged for an additional day. It would thus appear that the subunit