Comparison of ESR1 Mutations in Tumor Tissue and Matched Plasma Samples from Metastatic Breast Cancer Patients

BACKGROUND: ESR1 mutation in circulating cell-free DNA (cfDNA) is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. METHODS: ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC) (35 tumor tissue samples and 67 plasma samples). RESULTS: Of the 35 paired samples, 26 (74.3%) were concordant: one patient had detectable ESR1 mutations both plasma (ESR1 Y537S/Y537N) and tumor tissue (ESR1 Y537S/Y537C), and 25 had WT ESR1 alleles in both. Nine (25.7%) had discordance between the plasma and tissue results: five had mutations detected only in their tumor tissue (two Y537S, one Y537C, one D538G, and one Y537S/Y537N/D538G), and four had mutations detected only in their plasma (one Y537S, one Y537N, and two Y537S/Y537N/D538G). Furthermore, longitudinal plasma samples from 19 patients were used to assess changes in the presence of ESR1 mutations during treatment. Eleven patients had cfDNA ESR1 mutations over the course of treatment. A total of eight of 11 patients with MBC with cfDNA ESR1 mutations (72.7%) had the polyclonal mutations. CONCLUSION: We have shown the independent distribution of ESR1 mutations between plasma and tumor tissue in 35 patients with MBC.     

Actomyosin-like ATPase extracted from plain synaptic vesicle fractions

Ca²⁺-sensitive Mg²⁺-dependent ATP phosphohydrolase (EC 3.6.1.3, ATPase) was extracted from the plain synaptic vesicle fractions that were virtually devoid of contamination. The protein pattern of the ATPase preparation on SDS polyacrylamide gel electrophoresis closely resembled that of actomyosin from skeletal muscle. The finding suggests that the main components of the ATPase are actin- and myosin-like proteins of the brain (stenin and neurin). Microsome and synaptosomal plasmalemma fractions were extracted under the same conditions to examine the possibility that the ATPase extracted derived from contaminating particulates. An entirely different ATPase was extracted from microsomes, and no protein from plasma membranes. Although Ca²⁺-sensitive Mg²⁺-dependent ATPase was extracted from coated vesicle fraction, the electrophoretic pattern was dissimilar to that of the ATPase from plain synaptic vesicle fractions. It may be inferred that the whole complex of neurostenin is located in plain synaptic vesicles from the brain.     

Cellular events in protein-tolerant inbred rats. III. Antigen-reactive B cells in rats tolerant to human serum albumin.

Rats tolerant to human serum albumin (HSA) were injected with selected lymphocyte populations and challenged with HSA plus adjuvant to test for loss of tolerance. Thoracic duct lymphocytes (TDL) from normal or immune rats, either untreated or depleted of Ig-bearing cells or HSA-binding cells by affinity chromatography were all equally effective in restoring the HSA antibody response in previously tolerant recipients. In contrast, recirculating B cells (TDL from B rats) were not effective. The results indicated that unresponsiveness to HSA was a lesion of the T- but not the B-cell compartment. However, antibody affinity failed to mature to a high level in tolerant rats that were restored with T cells, and the response of transferred primed B cells into unresponsive recipients was inhibited, suggesting that the tolerant state was not merely due to a T-cell deletion.     

A rapid and simple method for the determination of base substitution and frameshift specificity of mutagens

A rapid method for the determination of mutagenic specificity has been developed which makes use of the ochre mutation (TAA) in the his-4 gene of Escherichiacoli. Reversion to His⁺ may occur by suppressor mutation (Type I) or by mutation within the his-4 gene (Type II). The Type I mutations may be further subdivided with respect to the type of suppressor mutation by their ability to suppress nonsense mutants of bacteriophage T4, thus allowing the identification of the responsible base substitution (Kato et al., 1980). The system has the ability to identify mutagens which produce A:T → G:C transitions since only Type II mutants can arise through this base substitution; and in fact, the system confirms the A:T → G:C specificity of the mutagen, N⁴-hydroxycytidine (Janion and Glickman, 1980) since only Type II mutants were induced by treatment with this base analogue.When this system was further tested with several additional mutagens, the results indicate that ethyl methanesulphonate, methyl nitrosourea and ethyl nitrosourea produce primarily Type I revertants which were primarily G:C → A:T transitions. UV-light, γ-rays, 4NQO and methyl methanesulphonate produced all types of base substitutions. The tester strain was further improved by introducing a series of sequenced trp^? frameshift mutations, thus allowing the simultaneous monitoring of frameshift and base-substitution mutations.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade.

Enzymes and regulatory proteins involved in the cascade control of glutamine synthetase activity of Escherichia coli have been separated from one another and the effects of numerous metabolites on each step in the cascade have been determined. The adenylyl transferase (ATase) -catalyzed adenylylation of glutamine synthetase, which requires the presence of the unmodified form of the regulatory protein PII is enhanced by glutamine and is inhibited by either α-ketoglutarate (α-KG) or the uridylylated form (PII·UMP) of the regulatory protein. PII·UMP and α-KG act synergistically to inhibit this activity. In contrast, the PII·UMP-dependent, ATase-catalyzed deadenylylation of glutamine synthetase requires α-KG and ATP and is inhibited by glutamine or PII and synergistically by glutamine plus PII. The capacity of uridylyl transferase (UTase) to catalyze the uridylylation of PII is dependent on the presence of α-KG and ATP and is inhibited by glutamine. The deuridylylation of PII·UMP by the uridylyl removing enzyme (UR) is enhanced by glutamine but is unaffected by α-KG. However, CMP, UMP, and CoA all inhibit activity at 10^?6m. High concentrations of ATase inhibit both UR and UTase activities, presumably by binding the regulatory protein. Of more than 50 substances that alter the activity of at least one enzyme in the cascade, only α-KG and glutamine affect the activity at every step. This accounts for the observation that glutamine synthetase activity in vivo is very sensitive to the intracellular ratio of α-KG to glutamine.     

Unusual metabolism of sulfur-containing amino acids in rats treated with DL-propargylglycine

S-(2-Hydroxy-2-carboxyethyl)homocysteine, S-(3-hydroxy-3-carboxy-n-propyl)-cysteine, N-acylated S-(beta-carboxyethyl)cysteine, and N-acylated S-(3-hydroxy-3-carboxy-n-propyl) cysteine were excreted in the urine after DL-propargylglycine treatment. Cystathionine was also accumulated in several tissues of DL-propargylglycine-treated rats. N-Monoacetylcystathione was found in the liver of rats and was also detected in the kidney and serum. Cystathionine gamma-lyase activity in liver decreased to about 4% of that of control rats 24 h after the DL-propargylglycine injection, and alanine aminotransferase activity decreased to about 35% of that of control rats. On the other hand, aspartate aminotransferase and cystathionine beta-synthese activity did not show significant changes from those of control rats. The ability of normal tissues to synthesize cystathionine utilizing cystathionine beta-synthase was 1.98 +/- 0.40 mumol/min/g in liver, 0.61 +/- 0.13 in kidney, and 0.18 +/- 0.015 in brain. The maximal contents of cystathionine in rat tissues and the administered amounts of DL-propargylglycine agreed well with the ability to synthesize cystathionine in each tissue.     

Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr⁺ or uvr^? strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His⁺, ColE^R and Spc^R, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N′-nitro-N-nitrosoguanidine-induced His⁺ mutations. In contrast, only 1 out of 13 trp^? frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC⁺-dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp⁺ mutations in most of the strains regardless of the umu⁺ or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC⁺-dependent pathway of error-prone repair.     

Induction of the differentiation of memory T killer cells with factors released from macrophage-like cell lines

Macrophage-like cell lines J774.1 and WEHI-3 as well as peritoneal exudate macrophages have been demonstrated to produce factors which induce the differentiation of memory cells into specific T killer cells in the absence of an added antigen. LPS stimulation was required for J774.1 cells and peritoneal macrophages to produce the factors but not for WEHI-3 cells. Interferon seemed to be one of the responsible factors. However, macrophages seem to produce other active factors; one has a molecular weight (MW) of more than 80,000 and lacks thymocyte mitogenic activity; another, with a weak thymocyte mitogenic activity, has a MW of 38,000 to 44,000. The low MW thymocyte mitogenic factor (interleukin-1) showed weak T killer cell differentiating activity.     

Serotonin involvement in the inhibition of luteinizing hormone (LH) release during immobilization in castrated male rats

The involvement of serotonin in mediating the inhibitory effect of immobilization stress on LH secretion in castrated male rats was examined by employing p-chlorophenylalanine (PCPA, 320 mg/kg, ip), an inhibitor of serotonin synthesis, and 5,6-dihydroxytryptamine (5,6-DHT, 50 micrograms, icv), a drug toxic to the indoleaminergic system. Immobilization stress suppressed pulsatile LH release and decreased mean plasma LH levels. Pretreatment with PCPA or 5,6-DHT apparently eliminated the inhibitory effect of immobilization stress on LH release. These results suggest the possible involvement of a serotoninergic mechanism in mediating the suppression of LH release induced by immobilization stress in castrated male rats.     

Isolation and characterization of mutator mutants from cultured mouse FM3A cells

A method to select mutator mutants was developed and 3 mutants were isolated from cultured mouse FM3A cells. Fluctuation analyses revealed that these mutator mutants have increased rates of spontaneous mutation at 3 genetic loci tested (resistance to ouabain, blasticidin S and tunicamycin). None of the 3 mutator mutants showed altered sensitivity to aphidicolin or arabinofuranosylcytosine, and so they differed from the mammalian mutator mutants reported previously. Also, all the mutator mutants had the same sensitivity as wild-type to UV or other DNA-damaging agents. Thus, these mutator mutants do not seem to have any deficiency in the DNA-repair process.

To determine whether the mutator activity was due to the intracellular dNTP pool imbalance, 4 dNTPs in these mutator mutants were determined by high-pressure liquid chromatography and compared to that of the wild-type cells. The results show that there is no large dNTP pool imbalance in these mutator mutants. Since the mutator activity is not associated with the dNTP pool imbalance, these mutants may have altered protein(s) directly involved in DNA replication.     

Changing behavior of surface immunoglobulin on mouse B lymphocytes during immune development: II. Surface immunoglobulin modulation clearly distinguishes two stages of B-cell maturation in the neonate

Antibody-induced modulation of cell surface IgM was used as a probe to explore membrane-related differences between fetal and adult mouse B lymphocytes. Receptor modulation was studied as a function of temperature in B cells obtained from mice of various ages. Plots of the rate of anti-IgM induced movement of receptors with temperature showed discontinuities (changes in slope) at different characteristic temperatures for each cell. In this way, radical differences between the receptor behavior of fetal B cells and adult B cells were evident. Moreover, temperature-dependent rates of internalization of receptor-ligand complexes also were found to clearly distinguish two stages of B lymphocyte maturation in the mouse.     

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow range ampholytes prepared in this way are comparable to commercial ampholyte preparation as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.     

Synergistic functions of phorbol ester and calcium in serotonin release from human platelets

In human platelets, thrombin activates Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) and mobilizes Ca2+ concomitantly, whereas 12-O-tetradecanoylphorbol-13-acetate (TPA) may be intercalated into membranes and directly activates protein kinase C without mobilization of Ca2+ in sufficient quantities. A series of experiments with TPA and Ca2+-ionophore (A23187) indicates that activation of protein kinase C is a prerequisite requirement for release of serotonin, and that this enzyme activation and Ca2+ mobilization act synergistically to elicit a full cellular response. Both cyclic AMP and cyclic GMP inhibit activation of protein kinase C by prohibiting the signal-dependent breakdown of inositol phospholipid to produce diacyl-glycerol, but none of these cyclic nucleotides prevents the TPA-induced activation of this enzyme.     

Asymmetric distribution of nitrate reductase subunits in the cytoplasmic membrane of Escherichia coli: evidence derived from surface labeling studies with transglutaminase.

The enzyme transglutaminase has been used to label surface proteins of Escherichia coli cytoplasmic membranes by covalently attaching to them a small fluorescent primary amine, dansyl cadaverine. Spheroplasts lacking outer membrane, osmotically lysed vesicles from the spheroplasts, and vesicles made by breaking cells in a French pressure cell were each labeled with transglutaminase and dansyl cadaverine. When the total cytoplasmic membrane proteins of each were examined on sodium dodecyl sulfate gels, three rather different labeling patterns were obtained. Labeling of the respiratory enzyme, nitrate reductase, in the membranes of each of these preparations was also examined. Membrane-bound nitrate reductase contains three subunits: A, B, and C. Dansyl cadaverine labeling of nitrate reductase in the presence of Triton X-100 indicated that subunits A and C could be labeled. When nitrate reductase was isolated from dansyl cadaverine-labeled spheroplasts, none of the subunits was labeled. When nitrate reductase was isolated from French press vesicles, subunit A was labeled and labeling was enhanced by the presence of nitrate during labeling. When nitrate reductase from osmotic vesicles was examined, subunit A was labeled in the presence of nitrate but no labeled subunits appeared when the vesicles were labeled in the absence of nitrate. It was concluded that (i) nitrate reductase is buried in the membrane with subunit A exposed only on the inner surface of the membrane, (ii) subunit C is sufficiently buried within the membrane so that it is inaccessible to transglutaminase, (iii) subunit B is not labeled under any condition, so its location is not known, and (iv) large osmotic vesicles are probably mosaics in which some protein components have been reoriented.     

An effect of diet on the activity of phosphofructokinase in rat heart

Phosphorylation of the inhibitory subunit of cardiac troponin (TN-I) occurs $\text{in vivo}$ after catecholamine intervention through adenylcyclase, cyclic AMP and cyclic AMP dependent protein kinase system. Also, TN-I and tropomyosin binding subunit of troponin (TN-T) are specifically hydrolyzed by calcium-activated neutral protease (CANP). In this study, we compared proteolysis of a set of TNs before and after phosphorylation by cyclic AMP dependent protein kinase plus cyclic AMP, using CANP from cardiac muscle. The initial rate of peptide release from both TNs was the same. After prolonged incubation, however, unphosphorylated TN degradation retarded, while phosphorylated TN proteolysis still continued. The amount of peptide release at the latter phase was dependent on the degree of phosphorylation. These results were confirmed by SDS polyacrylamide gel electrophoresis, and they suggest that a conformational change occurred in the whole TN molecule after phosphorylation of TN-I.     

Studies on heme d1 extracted from Pseudomonas aeruginosa nitrite reductase

Heme d1 has been extracted from Pseudomonas nitrite reductase. Imidazole, cyanide, and chloride-ferroheme, and CO, NO, cyanide, imidazole, and pyridine-ferroheme complexes have been prepared for study by UV/vis spectroscopy, and in some cass by epr and low-temperature mcd as well. Iron determinations have been carried out to assess extinction coefficients. Absorption spectra were used to monitor the transition of chloride-ferriheme d1 to an alkaline form of ferriheme d1 and a pka of 6.5 was determined for the process. The epr spectrum of chloride-ferriheme possessed the characteristic g = 6 signal of high spin (S = 5/2) iron, but the alkaline-ferriheme form gave no detectable epr signals. Electron paramagnetic resonance spectra were also obtained for cyanide and imidazole-ferriheme d1 and for NO-ferroheme d1. The imidazole complex gave signals that were very weak in comparison with the cyanide complex, but mcd measurements of imidazole-ferriheme d1 were consistent with it being a low-spin (S = 1/2) system. The epr signals of NO-ferroheme d1 were similar to those of the corresponding holo-enzyme complex. Reduction of alkaline-ferriheme d1 was found to be affected by the presence of oxygen, but under N2 give the same result with ascorbate and dithionite. Autoreduction of alkaline-ferriheme d1 was observed when placed under CO, and NO, atmospheres, or when treated with pyridine.