Mels Sluyser

Interaction of steroid hormones with histones in vitro

Abstract 1. [ 3 H]Hydrocortisone is bound more to F3 histone than to F1 or F2a histone in vitro . Aggregated F2a histone binds more [ 3 H]hydrocortisone than does unaggregated F2a histone. 2. The binding of [ 3 H]hydrocortisone to arginine-rich histone increases with the time of incubation. There is a linear increase of binding with increasing steroid concentration. The binding per mg histone decreases with increasing histone concentration. 3. Histone-bound [ 3 H]hydrocortisone is less easily removed from aqueous solution by extraction with chloroform, than is free [ 3 H]hydrocortisone. 4. When the histone-[ 3 H]hydrocortisone complex is added to a DNA solution, a large amount of steroid co-precipitates with the DNA-histone. 5. Evidence is presented to suggest that the hydrophobic region on the arginine-rich histone molecule has a relatively larger [ 3 H]hydrocortisone-binding capacity than the remainder of the molecule. 6. Arginine-rich histone which was oxidized with performic acid bound more [ 3 H]hydrocortisone than did untreated histone. 7. A model for the structure of arginine-rich histone and a possible mode of interaction of the arginine-rich histone with DNA, is tentatively proposed.

Role of estrogen receptor variants in the development of hormone resistance in breast cancer

Recent evidence suggests that the progression to hormone resistance in some breast tumors is due to mutations in the estrogen receptor (ER). Various types of ER variants have been found in breast cancer biopsies and breast cancer cell lines. The ER variants include dominant-positive receptors that are transcriptionally active in the absence of estrogen, and dominant-negative receptors that are themselves transcriptionally inactive but prevent the action of the normal receptor. The mechanisms by which these variants cause loss of hormonal control is becoming clear. ER variants may be prognostic factors for breast cancer. By modifying the action of ER variants, it should be possible to develop new strategies for treatment of malignant breast disease.

Studies on cytoplasmic ribonucleic acid from rat liver. I. Fractionation and function of soluble ribonucleic acid.

Abstract 1. 1. The soluble polynucleotides isolated by phenol extraction from rat-liver cytoplasm were fractionated on the resin Ecteola. 2. 2. Four fractions were obtained, one of which (s3-RNA) could be transferred to microsomal RNA in the presence of soluble enzymes, ATP, GTP and a nucleoside triphosphate regenerating system. The efficiency of this transfer was about 17% based on the isotope content of the added polynucleotides. 3. 3. On incubation of pH 5 enzymes with DL -[14C]leucine or a mixture of radioactive amino acids in the presence of ATP, s3-RNA became mainly labelled. Minor amounts of radioactivity, not exceeding 10% of that recovered in s3-RNA, were also detected in other fractions. 4. 4. The fractions were further characterized by ultracentrifugal analysis, dialysis and treatment with EDTA. 5. 5. The possible significance of these results for the mechanism of protein biosynthesis in rat-liver microsomes is discussed.

Estrogen responsive creatine kinase in normal and neoplastic cells.

Abstract Estrogen-responsive creatine kinase (uterine estrogen-induced protein, CK-BB) activity was compared during ontogeny of the rat uterus, in the human menstrual cycle and in mouse and human mammary tumors. Between days 2 and 26 of post-natal development of rat uterus, the specific activity of creatine kinase increases 4.5-fold. The glycolytic enzymes, phosphoglycerate kinase and phosphoglycerate mutase, show no increase and pyruvate kinase activity increases 1.5-fold, during this period. Only creatine kinase BB activity was increased by estrogen administration. In human endometrium, the specific activity of creatine kinase increases in the late-secretory stage of the menstrual cycle. During the progression from hormonal dependence to independence shown by the GR mouse mammary tumor in the course of successive transplantations, the total activity of creatine kinase increases. The increase in the MM (muscle type, non-estrogcn responsive) isozyme activity of creatine kinase exceeds the increase in creatine kinase BB activity. Normal human breast and breast tumors display both creatine kinase isozymes. Preliminary evidence for in vitro estrogen responsiveness of creatine kinase, in human breast explants, raises the possibility that creatine kinase BB may be a suitable marker for assessing the hormonal dependence of human tumors.

Studies on cytoplasmic ribonucleic acid from rat liver II. Fractionation and function of microsomal ribonucleic acid

Abstract 1. 1. Rat-liver microsomal RNA isolated by phenol extraction from deoxycholate-insoluble particles was fractionated on the resin Ecteola, yielding at least three fractions. 2. 2. Following in vitro incubation of microsomes with 32 P labelled soluble polynucleotides, AT ATP, GTP, and a nucleoside triphosphate regenerating system, only one microsomal RNA fraction became radioactive. 3. 3. This selective metabolic transfer of soluble RNA (or a derivative thereof) resulted in the attachment to microsomal RNA by covalent bonds. 4. 4. Ribonuclease activity in the ribonucleoprotein particles became manifest after exposure of the particles to 8 M urea. The presence of this enzyme is discussed in connection with the transfer reaction.

Inhibition of deoxyribonucleic acid synthesis in regenerating rat liver by the administration of histones in vivo.

Abstract 1. 1. The intraperitoneal injection of rat-liver preparations into partially hepatectomized rats inhibited DNA synthesis in the regenerating liver. Fractionation of the rat-liver preparations revealed that histone components were to a great extent responsible for the observed effects. 2. Injection of 10 mg polylysine also impaired DNA synthesis but equal doses of lysine or arginine had no effect. 3. Combinations of calf-thymus DNA with rat-liver basic proteins in vitro revealed that the lysine-rich histone fraction caused a much larger increase in the transition temperature (T m ) of DNA than any of the other fractions. In contrast, there was no significant difference between the specific inhibitory activities of the histone fractions in vivo .

Modulation of [5-125I]iododeoxyuridine incorporation into tumour and normal tissue DNA by methotrexate and thymidylate synthase inhibitors

A potentially useful method for imaging of micrometastases and in situ radiotherapy, would be the incorporation of radioactive labelled iododeoxyuridine (IdU) into tumour DNA. However, there are two main problems: incorporation of the radioactive IdU into normal cells and low incorporation into tumour cells. The aim of this study was to attempt to augment the incorporation of [5-125I]iododeoxyuridine (125IdU) into tumour DNA and to improve the tumour/normal tissue ratio by the use of inhibitors (methotrexate, 5-fluorouracil, AG337, ZD 1694, benzyloxybenzyl uracil) which would prolong the metabolic half-life of the compound. Mammary tumours were induced in GR mice, which were then treated with the inhibitors and the 125IdU. The tumours and representative normal tissue were removed following sacrifice of the animals, and radioactivity within the tissues measured. Pretreatment of mammary carcinoma-bearing GR mice with methotrexate caused approximately a 3-fold increase in the incorporation of 125IdU into tumour DNA, and approximately a > or = 10-fold increase in the tumour/small intestine ratio of incorporated radioactivity. Inhibition of thymidylate synthase, the enzyme involved in IdU dehalogenation, by 5-fluorouracil plus folic acid, or by novel inhibitors AG337 and ZD1694 led to a 3- to 5-fold increase in the 125IdU incorporation. Benzyloxybenzyl uracil, an inhibitor of dihydrouracil dehydrogenase, had little effect. Treatment of tumour-bearing mice with methotrexate plus ZD1694 significantly reduced the rate of tumour growth, but addition of 125IdU (70 microCi/mouse, three daily injections) had no additional antitumour activity. In conclusion, these results do not support the hypothesis that systemic administration of 125IdU can be used for cancer therapy or for imaging purposes unless better methods are found to boost its incorporation into tumour DNA.

Interaction of steroid hormone receptors with DNA

Abstract Recent investigations suggest that steroid hormone receptors recognize specific binding sites on nuclear DNA, and that A + T rich regions may be involved. A model is proposed by which this interaction may take place.

Metabolism of a [18F]fluorine labeled progestin (21 -[18F]fluoro-16α -ethyl-19-norprogesterone) in humans: a clue for future investigations

Assessment of estrogen receptors and progesterone receptors (PR) with PET may allow the determination of the hormone responsiveness of tumors without the need for multiple biopsies, and the monitoring of the effect of hormonal therapy. In spite of the favourable characteristics of 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone ([18F]FENP) found in preclinical studies, the compound failed to reveal the presence of PR in breast carcinomas and meningiomas. In view of the clinical significance of the PR assay in human breast cancer, it is worthwhile to explore mechanisms that are potentially involved in the inadequacy of [18F]FENP to image PR with PET. Our present study on the in vivo metabolism of [18F]FENP in humans demonstrates a rapid clearance and biotransformation of the compound. Results of incubation experiments suggest that the metabolic conversion of [18F]FENP is not restricted to the liver, but also occurs in blood cells (presumably the erythrocytes) and tumors (breast carcinomas and meningiomas). The predominant metabolite of [18F]FENP in plasma during the rapid distribution phase and in tumors is identified as 20-dihydro-[18F]FENP. The conversion of [18F]FENP to its 20 alpha- or 20 beta-hydroxy metabolite has a deleterious effect on the binding affinity for PR. Our findings do not justify a conclusion as to the extent of in vivo extrahepatic biotransformation of [18F]FENP, or its significance in the ineffectiveness of [18F]FENP as an imaging agent for PR. On the other hand, the ability of breast carcinomas and meningiomas to metabolize [18F]FENP avidly appears to preclude selective imaging of PR in these tumors during the time of a PET examination. It is imperative to evaluate the metabolic stability of a [18F]fluorine labeled progestin in an early stage of future screening procedures.

Antigenic determinants on lysine-rich histones.

Abstract 1. 1. Rabbit sera were prepared containing antibodies which reacted specifically with lysine-rich histones. Immunofluorescent studies showed specific staining of cell nuclei with these antisera after appropriate absorption of the sera with non-histone proteins. 2. 2. Complement fixation studies revealed that lysine-rich histones from rat tissues contain antigenic determinants common with other species and also rat-specific antigenic determinants. When the amino-terminal region (amino acid residues 1–73) was detached from the lysine-rich rat liver histone, the remaining fragment still contained both types of antigenic determinants. 3. 3. The antisera could not distinguish between lysine-rich histones from normal rat tissues and those from rat tumors.