Biomedical subjects
Publications and source records attributed to C W Porter.
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Methylglyoxal-bis(guanylhydrazone) (MGBG) is a polycationic drug which is useful in the chemotherapy of lymphoid and myeloid proliferative disorders. The drug has recently been shown to produce selective ultrastructural damage to the mitochondria of proliferating cell populations. It is important to understand the molecular basis for this action, since it may be related to the known ability of MGBG to block polyamine biosynthesis. Accordingly, the effect of MGBG treatment on the incorporation of [3H]thymidine into both mitochondrial and nuclear DNA has been examined. Exponentially growing L1210 leukemia cells were prelabeled with [14C]thymidine, treated with MGBG for 1.5 to 16 hr, and then pulse labeled with [3H]-thymidine. Incorporation of [3H]thymidine into mitochondrial DNA was selectively inhibited at 5 hr with concentrations of 1 to 10 microM MGBG. Nuclear DNA, however, was not similarly affected until 8 to 11 hr of drug treatment. Dye-CsCl gradients of mitochondrial DNA indicated that the inhibition of synthesis occurred in replicative forms of circular DNA. Uptake studies excluded the possibility of drug interference with cellular uptake of thymidine. Ultrastructural studies revealed a very close correlation between the dose-response curve for mitochondrial damage and that for MGBG inhibition of mitochondrial DNA synthesis. This correlation suggests a direct cause-and-effect relationship between inhibition of mitochondrial DNA synthesis and ultrastructural damage, but the possibility of both phenomena being related to another action by the drug, such as inhibition of polyamine biosynthesis, or a drug effect on mitochondrial function, must also be considered.
The suitability of electron microscope autoradiography for sutdying the uptake and intracellular localization of lipid vesicles (liposomes) containing radiolabeled saturated phospholipids has been examined. Data are presented showing that preparation of specimens for electron microscope autoradiography by conventional methods is accompanied by significant translocation and intercellular redistribution of radiolabeled saturated lipids, causing spurious labeling patterns. Intercellular redistribution of radiolabeled lipid was demonstrated by mixing glutaraldehyde-fixed mous L1210 cells that had been incubated with sonicated lipid vesicles containing [H] dipalmitoyl phosphatidylcholine with an indicator cell population (fixed avian erythrocytes) which had not been exposed to vesicles and showing that after electron microscope processing radiolabeled grains were present in both cell types. The same redistribution artifact also probably affects the intracellular localization of radiolabeled lipids. This artifact is discussed in relation to previous work in which autoradiographic methods have been used for ultrastructural localization saturated phospholipids in cells and tissues.
The proliferation kinetics of a fast-growing spontaneous mouse mammary tumor subline (SMT-F) and a slow-growing spontaneous mouse mammary tumor subline (SMT-S) tumor have been determined autoradiographically at 2 different stages of tumor growth. The length of the cell cycle and the growth fraction for SMT-F were 11.2 hr and 0.85, respectively, on Day 14 and 12.1 hr and 0.78 on Day 28. For SMT-S these same parameters were 15.6 hr and 0.50 on Day 14 and 16. 1 hr and 0.45 on Day 28. On Days 14 and 28 the mitotic indices were 1.3 and 1.0%, respectively, for SMT-S, compared to 2.2 and 1.9% for SMT-F. The cell loss rate, cell loss factor, and cell loss were significantly higher for SMT-F than for SMT-S. The difference in the growth rates for these 2 tumor lines was attributable to a slight prolongation of the length of the cell cycle and a reduction in the growth fraction of SMT-S.
Exposure of cultured leukemia L1210 cells to 0.1 micron methylglyoxal-bis(guanylhydrazone) resulted in a concentration-dependent inhibition of cellular proliferation, beginning after about 1 to 2 generation times (12 to 24 hr). Ultrastructural examination of the cells treated at and above 1.0 micron concentrations of drug for 24 hr revealed unifrom damage to mitochondria. The basic lesion involved extensive swelling of the mitochondrion, a deterioration and eventual loss of cristae, and a decrease in the matrix density. This damage preceded growth inhibition by about 12 hr and did not immediately affect cell viability as detected by trypan blue dye exclusion. Whether these findings are related to the known actions of the drug on polyamine metabolism is not clear at present.
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The acetylcholine receptors at the mammalian motor end plate have been counted and their distribution there determined ultrastructurally. Electron microscope autoradiography applied to muscles labeled with alpha-(3H)bungarotoxin was used for this purpose. The receptors are distributed asymmetrically along the postsynaptic membrane, being concentrated at the fold crests-that portion nearest to the presynaptic membrane. The density of receptor sites at that region is estimated to be 20,000-25,000 per mum2 of membrane surface. This density holds for the several species and muscle types thus far examined and appears to be a constant parameter of the motor end plate. It determines the limit of responsiveness to acetylcholine. By contrast, the enzyme acetylcholinesterase is found to be distributed evenly over the folds and may reside in the intersynaptic matrix. When mouse diaphragm end plates from dystrophic animals or animals following 5-day denervation were similarly examined, no significant alterations in either the density or the distribution of the receptor sites were found. Similarly, dystrophic muscles in chickens possess an unaltered number of receptors at their end plates. A model is outlined to correlate receptor and cholinesterase concentrations with known aspects of transmitter release. These findings may have relevance to some of the electrophysiological abnormalities seen in myasthenia gravis.
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Electron-microscope autoradiography of diaphragm endplates of the American brown bat, labeled to saturation with tritiated alpha-bungarotoxin, has been used as a means to localize and quantitate the acetylcholine receptor there. Analysis of the grain distribution in these autoradiographs reveals that the receptor sites in this endplate are located on the postsynaptic membrane at an average density of 8,800/mu2. The sites are distributed asymmetrically along that membrane, being concentrated at the crests of the postjunctional folds--that portion nearest to the presynaptic membrane. The receptor site density at these regions of the postsynaptic membrane is estimated to be 20,000--25,000/mu2 of membrane surface. A comparison of these membrane site densities with those of endplates of red and white fibers of the mouse reveals a close similarity. On this basis, it is suggested that the receptor site density at the crests of the folds may be a characteristic feature of endplates of vertebrates. In contrast to the acetylcholine receptor sites, cholinesterase sites (determined autoradiographically in 3H-diisopropylfluorophosphate-labeled endplates) are largely distributed in a uniform manner over the postjunctional folds. The function of the secondary folds is, therefore, reassessed. Ultrastructural evidence available from other laboratories on the spatial characteristics of transmitter release and of postsynaptic dense particles is in accord with a model drawn for this molecular architecture at the vertebrate endplate.
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In various skeletal muscles, the mean density of acetylcholine receptors in the muscle postsynaptic membrane is constant at about 8700 per mum(2), even though the overall size of an endplate ranged from 400 mum(2) to 1300 mum(2) in these muscles. This measurement was by electron microscope autoradiography of alpha-[(3)H]bungarotoxin binding sites; only one-half of these, however, appear to be true active centers of the acetylcholine receptor. The highest density of these receptors is on the juxtaneuronal regions of the postsynaptic membrane, and their density in the depths of the fold is less than one quarter of that at the tips. A maximum sensitivity to externally applied acetylcholine, about 3000-5000 mV/nC, is found in diverse types of endplates when truly focal recording is achieved. This acetylcholine sensitivity appears to be determined by the local density of receptors in the membrane, and not by their total number at the endplate. A quantal efficiency term is also disclosed. The maximal sensitivity per molecule obtainable by microiontophoresis of acetylcholine is 5-10% of that operative when a quantum reacts. When acetylcholine is released from a vesicle, in contrast to its application from outside, geometric factors are more favorable. Consideration of the local packing density, acetylcholine molecule numbers, and the current flow when one quantum of acetylcholine interacts at the membrane suggests that one, or possibly two, activated receptor active centers are linked to one open gate of the ionic conductance modulator.
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We have previously found that in one of two human melanoma cell lines, potent increase in the polyamine catabolizing enzyme, spermidine/spermine N1-acetyltransferase (SSAT), correlate with growth sensitivity to the spermine analog, N1, N12-bis(ethyl) spermine (BESPM). Herein, we examine the generality of this SSAT response among seven human melanoma cell lines (LOX, SH-1, STO-1, HO, PANUT-3, MALME-3 and Ebey) and normal melanocytes and further evaluate its possible correlation with growth sensitivity. Following treatment with 10 microM BESPM for 48 hr, SSAT activity among the various cell lines increased from basal levels of 20-90 pmol/min/mg to levels ranging from 170 to 30,470 pmol/min/mg. Five of the seven cell lines and melanocytes induced SSAT activity to levels to greater than 2,500 pmol/min/mg and three of these, to levels greater than 10,000 pmol/min/mg. When ranked according to SSAT responsiveness (LOX less than SH-1 less than STO-1 less than HO less than PANUT-3 less than MALME3 less than Ebey), there was a general correlation among the cell lines with growth sensitivity. Antiproliferative effects ranged from slowing of cell growth in the less SSAT responsive lines (LOX, SH-1) to total cessation of cell growth or overt cytotoxicity in the more potently SSAT responsive lines (MALME-3, Ebey). The polyamine biosynthetic enzyme activities, ornithine and S-adenosylmethionine decarboxylase, were similarly suppressed in all cell lines, presumably via analog activation of inherent regulatory mechanisms. Polyamine pool reduction occurred to a greater extent than predicted in cell lines where SSAT was increased to greater than 2500 pmol/min/mg suggesting a possible role for the enzyme in enhancing polyamine excretion and/or catabolism. The occurrence of potent SSAT induction among several human melanoma cell lines and the growth sensitivity of these same lines to BESPM suggests that the enzyme response may represent a determinant of drug action in this particular malignancy.