Vincent P. Hollander

A simplified assay for RNase activity in crude tissue extracts

Abstract A method is described for the precipitation of unhydrolyzed RNA after RNase digestion that avoids the use of strong acids and is extremely sensitive for enzyme assay. The dilution step usually required for spectrophotometric measurements of supernatant fractions after separation from unhydrolyzed RNA is eliminated. The precipitating reagent is buffered lanthanum-magnesium-ethanol added in 10-fold volume to enzyme incubation mixtures. In the macroassay, an increase in absorbancy of 0.41 is obtained with 1 mμg bovine pancreatic RNase after 15 min incubation at 37°. For micromethods, the ethanol can be replaced by Cellosolve (2-ethoxyethanol) and the sensitivity increased 5- to 10-fold.

A sensitive spectrophotometric method for measurement of plasma endotoxin.

Abstract A spectrophotometric method for the measurement of 10−1–10−4 ng/ml of bacterial endotoxin in plasma is described which depends on the reaction of endotoxin with Limulus crab lysate and the removal from plasma of an inhibitor to the Limulus system by Bio-Gel P-200 filtration. Sensitivity is achieved by recorder scale expansion and by a linear transformation of data.

Role of nucleoside transport in glucocorticoid-induced regression of mouse lymphoma P1798

Abstract Exposure of corticoid-sensitive P1798 lymphocytes to cortisol results in inhibition of uridine uptake and incorporation with no effect on 2-deoxyglucose transport. Nucleoside uptake by corticoid-resistant cells is not affected by the hormone. Inhibition of uridine transport may play a key role in tumor regression and does not depend on reduced availability of glucose.

A rapid radiochemical assay for hypoxanthine-guanine phosphoribosyltransferase☆

Abstract A simple radiochemical method is described for assay of hypoxanthine-guanine phosphoribosyltransferase. 14C-Hypoxanthine is incubated with enzyme PRPP. The labelled product is precipitated on strips of Whatman No. 1 paper by the addition of lanthanum nitrate. Unreacted substrate is eluted with distilled water. The major advantages of this method are speed, reproducibility, ability to process many samples and low blank values.

Development of a somatotropic variant of the mammosomatotropic tumor MtT-W5.

Summary Repeated resection and reimplantation of the pituitary tumor MtT/W5 in W/Fu rats leads to the appearance of metastatic tumor chiefly in the ovaries and long bones. Metastatic tumor begins to appear about 15 weeks after initial subcutaneous implantation. Subtransplantation of some of the metastatic tumors results in a number of stable somatotropic tumors devoid of mammotropic activity. Whether this change in secretory activity is due to cloning or transformation cannot be determined at this time.

Difference in the Number of Insulin Binding Sites between Cortisol-Sensitive and Cortisol-Resistant Lymphoma P1798 Cells

Summary Cortisol-sensitive and cortisol-resistant lymphoma PI798 cells specifically bind [125I]insulin. Resistant lymphocytes bind 40% less insulin than sensitive cells. These results suggest that insulin (or insulin-like substances) may play a role in growth regulation and/or response of this tumor to glucocorticoid therapy.

The Correlation between Plasma Cell Tumor Development and Antibody Response in Inbred Strains of Mice

Summary The antibody response to bovine serum albumin and the development of hyperglobulinemia following administration of intraperitoneal mineral oil separated BALB/c and A-mice from DBA, C57B1, and C3H mice. BALB/c mice developed many plasma cell tumors following intraperitoneal mineral oil injections. The A-mice developed 1/10 tumors but the peritoneal contents showed extensive phagocytosis of tumor cells. This phenomenon is observed in BALB/c mice only when tumor development is inhibited by administration of glycoprotein pituitary hormones. A hypothesis is developed to explain these observations.

Estrogen receptor can distinguish among various halodeoxyuridine-substituted DNAs

Although the interplay between steroid hormone receptors and the genome has been extensively studied [l-6] , it is not yet known how the receptor protein interacts with double-stranded helical DNA. To examine the specificity and the dynamics of DNA-estrogen receptor (ER) interactions, we have initiated a study to determine how specific changes in the DNA will affect receptor binding. We have found that ER binds preferentially to AT-rich DNA [7] and also to DNA substituted with bromodeoxyuridine (BrdUrd) [8] . Numerous studies, in a wide spectrum of cell systems, have examined various changes in gene expression which follow BrdUrd-substitution in the DNA [9,10] . Relatively few experiments, however, have focused on the possible mechanism(s) by which this defined replacement (of the S-methyl group in thymine by a bromide atom) might cause subtle electronic or steric effects in DNA which, in turn, might alter gene expression in the eukaryotic cell [g-12]. One such mechanism involves altered binding of regulatory proteins to BrdUrd-substituted DNA [g-12]. One approach to the question of how the bromine atom causes its particular effects, is to ask if other halogen atoms in the same position cause the same effects [ 121. Therefore, we have incorporated 5-chloro-, 5-bromoand Siodo-deoxyuridine into DNA in equivalent molar amounts and determined the effect of these substitutions on the ER-

Effect of 9α-fluoroprednisolone and l-asparaginase on uridine incorporation into ribosomal RNA of P1798 lymphosarcoma

Abstract The effect of the lympholytic agents, 9α-fluoroprednisolone and l -asparaginase ( l -asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli (enzyme 2) on the incorporation of uridine into ribosomal RNA (rRNA) of P1798 lymphosarcoma was studied. After a 6- or 18-h treatment with either 9α-fluoroprednisolone or l -asparaginase, there was a marked decrease in incorporation into 18-S and 28-S rRNA. Incorporation into the 28-S component was significantly decreased 3 h after the administration of l -asparaginase, whereas 9α-fluoroprednisolone had no effect at this time. Neither 9α-fluoroprednisolone nor l -asparaginase depressed precursor incorporation in mice bearing steroid- or asparaginase-resistant tumors. These results suggest that changes in rRNA metabolism are related to tumor regression.

Study of the Photoattachment of Estrogen Receptor to the Nuclear Acceptor Sites in Human Breast Cancer Cells

A model for estrogen hormone action has been in vogue for over a decade.’.’ For example, the steroid binds to a specific cytoplasmic receptor and the hormone receptor-complex then migrates to the nucleus, where it presumably binds to chromatin to trigger off a specific alteration in gene expression.’-5 Precisely what happens in the nucleus is still unknown. Although the nature of specific interactions between estrogen receptors and the genetic material has been extensively ~tudied,’.~ there is, as yet, no evidence for direct contact between the receptor and the DNA in the target cell nucleus. I f there is an intimate contact between the receptor protein and DNA, and if we introduce a short cross-link between the protein and DNA, then the position of this cross-link will identify the point of contact. Our approach to this problem was to study the photochemical attachment of estrogen-receptor complex to bromouracil-substituted DNA (photochemically reactive DNA) in the intact nucleus of human breast cancer cells, MCF-7. We selected MCF-7 cells because they are estrogen responsive and contain estrogen receptor, which can translocate from the cytosol to the nucIeus.’ Our experiment was designed to follow the effect of irradiation on the extractability of the nuclear receptor as follows: I . Cells were grown in the presence of bromouracil, so that the bromouracil was incorporated into the nuclear DNA 2. The bromouracil-substituted cells were labeled with ’H-estradiol, so that the cytoplasmic receptor complex translocated to the nucleus 3. The cells were irradiated with near-uv light (-312 nm) to form a covalent cross-link between the receptor and the DNA