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R Hilf

Publications and source records attributed to R Hilf.

At least 109 records · Page 6Linked to original sources
PubMed1983

Effect of environmental hydrogen ion concentration on regulation of insulin receptors in cultured R3230AC mammary carcinoma cells.

Insulin binding and responsiveness in primary cultures of R3230AC rat mammary tumor cells were studied as a function of the hydrogen ion concentration of the culture medium. When insulin binding was assayed at pH 7.4, cultures that were maintained for a sufficient length of time in acidic medium demonstrated a significant increase in insulin receptor concentration compared to control cultures. The increase in insulin binding, which occurred as cultures approached confluency, was attributed to the increased acidity rather than nutrient depletion of the culture medium. In contrast, maximum binding was observed when the pH of the assay buffer was above 8.0, independent of the culture conditions. Binding of Concanavalin A, reflecting more generalized cell surface glycoproteins, decreased as cultures approached confluency, but was unaffected by the pH of either the culture medium or the assay buffer. The effect of pH on insulin responsiveness was studied. Insulin receptors generated by acidic culture conditions demonstrated insulin-induced down-regulation. Regardless of the pH environment, all cells demonstrated the same amount of insulin binding after exposure to 10(-6) M insulin. Under the in vitro conditions employed, cultured cells did not demonstrate a significant response to added insulin by alteration in growth, substrate transport, or incorporation of precursors into macromolecules, although the basal rates of these parameters were lower in cells maintained in acidic pH environments. The data presented indicate the necessity of considering the pH of the culture medium in studies of receptor regulation. It is possible that tumor cells, due to increased lactic acid production, may be especially prone to these changes.

3-O-Methylglucose↗
PubMed1983

Photosensitization of mitochondrial cytochrome c oxidase by hematoporphyrin derivative and related porphyrins in vitro and in vivo.

Mitochondrial cytochrome c oxidase activity was inhibited by exposure to hematoporphyrin derivative followed by photoirradiation. Inhibition of enzyme activity in vitro was a dose- and time-related event, the log of percentage inhibition being linear with time. Exposure of mitochondria to hematoporphyrin or hydroxyethylvinyldeuteroporphyrin sensitized mitochondria to photoirradiation, whereas protoporphyrin IX was only weakly active as a photosensitizer for inhibition of cytochrome c oxidase. Mitochondria from mammary glands of pregnant rats showed hematoporphyrin-induced photosensitivity similar to those from R3230AC mammary adenocarcinomas. Mitochondria prepared from tumors of animals given injections of hematoporphyrin derivative in vivo and then photoirradiated in vitro demonstrated considerable sensitivity to light as reflected by significant inhibition of cytochrome c oxidase activity. A side by side comparison of hematoporphyrin derivative with hematoporphyrin, using this in vivo-in vitro experimental protocol, indicated that photosensitivity was retained for a longer time after treatment with hematoporphyrin derivative. Taken together, these data provide a potential mechanism of action, i.e., inhibition of respiration, for porphyrin-induced photosensitivity, and they offer a useful assay to investigate this family of therapeutic agents.

Animals↗
PubMed1982

A logistic model based on enzyme activities for the prediction of response of breast cancer patients to chemotherapy.

A logistic regression model, utilizing the activities of certain selected glycolytic enzymes and ER status measured on primary or recurrent lesions, has been applied to predict for response to combination chemotherapy regimens administered to women with advanced breast cancer. The clinical outcome of response or no response was evaluated retrospectively using criteria employed by cooperative group protocols. In 93 cases, 58/61 patients classified as nonresponders and 22/32 patients demonstrating objective responses would have been correctly designated, based on the 50% estimated probability as the level for separation of responders from nonresponders. The overall predictive accuracy of this model was 86%, with apparently greater accuracy for prediction of lack of response. Addition of estrogen receptor status to the model imparted no gain in accuracy of prediction. Application of this model to a prospective study is warranted.

Adult↗
PubMed1982

Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma.

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of 125I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, Ke. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for Kd. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.

Adenocarcinoma↗
PubMed1982

Reduction of proline transport in R3230AC mammary carcinomas by estrogens in vitro.

The effects of estrogens on proline and leucine transport into enzymatically dissociated cells from the R3230AC mammary adenocarcinoma were studied. Estradiol-17beta demonstrated a time- and dose-dependent reduction of proline transport; at 10-6M, transport of proline was decreased by 50 percent. Kinetic analysis of these effects indicate that estradiol displayed characteristics of a non-competitive inhibitor, with a Ki of 1.79 microM. Other estrogens, and the anti-estrogen tamoxifen, gave somewhat higher estimated Ki values and could be ranked as inhibitors as follows estradiol 17beta greater than diethylstilbestrol greater than tamoxifen greater than estriol congruent to estrone greater than estradiol-17alpha. No effects of these estrogens on leucine transport were observed, indicating their selectiveness for the A system. The synthetic glucocorticoid, dexamethasone, was comparable to diethylstilbestrol as an inhibitors of proline transport, but dexamethasone also decreased transport of leucine, Testosterone and progesterone were approximately comparable to estrone in their actions. It is proposed that these actions of estrogens represent one potential mechanism whereby pharmacological levels exert therapeutic benefit in the treatment of advanced breast cancer.

Adenocarcinoma↗
PubMed1982

Down-regulation of insulin receptors in primary cultures of R3230 AC rat mammary adenocarcinoma cells.

Binding of insulin and Concanavalin A to primary cell cultures of the R3230AC rat mammary adenocarcinoma was studied as a function of time in culture. As the culture became confluent, the amount of insulin binding per cell increased with culture time and reached a plateau, whereas the binding of Con A to surface glycoproteins decreased to 50% of the initial value. Exposure of confluent cultures to insulin at 37 C resulted in down-regulation of the cell surface insulin receptors. The decrease in insulin binding was related to the ambient insulin concentration and the decreased numbers of receptors per cell with no apparent alteration in their affinity. The maximal decrease in receptor number was 60-70%. Cell cultures degraded significant amounts of insulin at 37 C, but the addition of bacitracin to the culture medium decreased the amount of degradation and increased the extent of down-regulation at each insulin concentration. Porcine proinsulin was less effective than insulin in competing with 125I-labeled insulin and inducing receptor down-regulation. Down-regulation of insulin receptors did not require protein synthesis. The rate of insulin-induced receptor loss was much faster than the decrease in insulin binding due to inhibition of protein synthesis by cyclohexamide. The estimated half-life of the insulin receptor was 10.5 h. Down-regulation of insulin receptors was reversible; regeneration of receptors to 50% of control levels occurred approximately 9.6 h after the removal of insulin and required protein synthesis. These results indicate that these mammary tumor cells retain the ability to regulate their insulin receptors.

Adenocarcinoma↗
PubMed1981

The hormonal regulation and interaction of concanavalin A and insulin binding in R3230AC mammary adenocarcinoma cells.

To investigate the effects of concanavalin A on insulin binding to R3230AC mammary carcinomas, initial experiments were performed to characterize binding of concanavalin A. Concanavalin A binding was found to be specific and saturable. Equilibrium binding experiments demonstrated that addition of low concentrations of concanavalin A enhanced the binding of [3H]concanavalin A, suggestive of positively cooperative interactions. Binding of concanavalin A was responsive to hormonal alterations; tumor cells from diabetic rats showed enhanced binding of concanavalin A and insulin compared to cells from intact rats and administration of insulin to diabetic rats returned concanavalin A and insulin binding to levels seen in controls. Incubation of tumor cells with concanavalin A prior to addition of 125I-labelled insulin resulted in a reduction of insulin-binding capacity; succinyl-concanavalin A did not affect binding of insulin. The present inhibition of insulin binding by concanavalin A was highest at the lower insulin concentrations, providing a linearized Scatchard plot that yielded a calculated Kd value comparable to the low-affinity portion of the curvilinear Scatchard plot for insulin binding. The dissociation rate of bound insulin depended on receptor occupancy. Addition of concanavalin A after insulin binding reached equilibrium resulted in increased insulin binding at higher hormone concentrations, decreased rates of dissociation of insulin and a loss of the correlation between receptor occupancy and dissociation rates. Concanavalin A alone demonstrated an insulin-like effect on glucose transport, which in these tumor cells represents a decrease in transport of 3-O-methylglucose. These results suggest that binding of both concanavalin A and insulin to cells from this hormonally responsive neoplasm is under insulin regulation and demonstrates similar characteristics to those reported for a variety of normal cells. Furthermore, the interaction between concanavalin A and te cell membranes affects the affinity of the insulin receptor for insulin and appears to decrease the observed negative cooperativity.

3-O-Methylglucose↗
PubMed1981

Low tissue enzyme activity seen in breast cancers of patients who fail adjuvant chemotherapy.

Primary breast cancers from 54 patients undergoing postsurgical adjuvant chemotherapy were analyzed for several enzymes and estrogen receptors to determine if biochemical parameters might offer a prognostic index for determining outcome of therapy. Recurrence of disease during therapy or within 12 months after cessation of therapy was the criterion for classifying the patient as a treatment failure. Recurrence rates were similar in ER-positive vs. ER-negative patients, indicating that estrogen receptor status offered no predictive value in this series. However, patients who failed therapy had tumors that displayed significantly lower activities of lactate dehydrogenase, glucosephosphate isomerase, and glucose 6-phosphate dehydrogenase vs. patients who showed no evidence of recurrence for this observation period. When either premenopausal or ER-negative patients were compared, a similar observation was made; low enzyme activities occurred in patients failing to benefit from adjuvant chemotherapy. Based on this series of patients, it is suggested that a selected enzyme activity profile may offer a predictive index for recurrence in the adjuvant chemotherapy setting.

Breast Neoplasms↗
PubMed1981

Effect of estrogen on metabolism and enzyme activities of mammary adenocarcinoma R3230AC in Fischer rats.

Metabolic characteristics and enzyme activities are reported for a variant line of R3230AC tumor, a long-established, transplantable mammary adenocarcinoma in the Fischer rat. Lipogenic enzyme activities from the variant tumor are generally lower than those reported for early generations of this tumor. In addition, the proportion of the medium-chain-length fatty acids in the lipids of this variant line is lower. In the variant tumor, estrogen treatment of host animals results in a reduction of fatty acid synthesis from both acetate and glucose. The response of the variant tumor to estrogen treatment is similar to that for the normal gland in a virgin rat in that lipogenic enzyme activities are lowered in both normal and neoplastic tissues. These enzymatic activities in early generations of tumor R3230AC were similar to those observed in mammary glands from lactating rather than virgin rats. Since the variant tumor still responds to estrogen treatment by a reduced growth rate, we have inferred that, although it undergoes the same structural differentiation observed in earlier generations, the variant tumor line no longer shows certain biochemical changes associated with the lactational state in the normal gland.

Adenocarcinoma↗
PubMed1980

The lack of relationship between estrogen receptor status and response to chemotherapy.

The possible relationship between estrogen receptors and response of breast cancer patients to chemotherapy was examined in two situations: patients with advanced disease and patients receiving post-surgical adjuvant treatment. In 73 patients with disseminated disease, no relationship between ER status and response to cytotoxic chemotherapy was observed. At this time, 44 of these cases have undergone extramural review; in these 44 cases, we observed no significant relationship between ER status and response to chemotherapy. In a series of 52 patients receiving adjuvant therapy, 27 patients were classified as failures due to recurrence. ER status did not offer prognostic value for failure of patients treated with postsurgical adjuvant therapy. We conclude that the prognostic value of ER data in nonhormonal therapy settings remains to be proven

Antineoplastic Agents↗
PubMed1980

The relative importance of estrogen receptor analysis as a prognostic factor for recurrence or response to chemotherapy in women with breast cancer.

The value of estrogen receptor (ER) analysis in primary breast cancer samples as a potential prognostic factor was examined in three clinical situations: time to recurrence in patients with no therapy after mastectomy, failure of patients receiving adjuvant therapy, and response of advanced disease patients to cytotoxic chemotherapy. Other prognostic factors analyzed were menopausal and nodal status. In none of these clinical settings were we able to demonstrate the usefulness of ER status as a prognosticator of the disease course or its response to therapy.

Antineoplastic Agents↗
PubMed1980

Influence of proliferative rates and A system substrate availability on proline transport in primary cell cultures of the R3230AC mammary tumor.

Regulation of A system amino acid transport was studied in primary cultures of the R3230AC mammary adenocarcinoma. Higher rates of carrier-mediated Na+-dependent proline transport, vc, was decreased and was attributed to a two-fold decrease in Vmax and a two-fold increase in Km. When compared to cells grown in standard media (Eagle's minimal essential medium, MEM), cells grown in media supplemented with A system substrates (alanine, serine, glycine, and proline) demonstrated adaptive decreases in proline transport; the decrease was due to two-fold reduction in Vmax, with no change in Km for proline. Even in the presence of preferred substrates for the A system, a density-dependent decrease in proline transport was manifested. Both fast- and slow-growing cultures maintained in MEM exhibited rapid increases in proline transport when switched to buffers devoid of amino acids; two-fold increases in Vmax were seen within 4 hr, but Km was unchanged. This starvation-induced adaptation was completely prevented by inclusion in the buffer of 10 mM proline, 0.1 mM alpha-(methylamino)-isobutyric acid (MetAIB) or 10 mM serine, whereas inclusion of the poorer A system substrate, phenylalanine (10 mM), had no effect. The effects of MetAIB to prevent starvation-induced increases in proline transport were dose-related, rapid, and reversible. Amino acid starvation-induced increases in proline transport were partially blocked by cycloheximide or actinomycin D. Data were obtained demonstrating a temporal relationship between increasing intracellular [proline] and decreasing vc for proline uptake. In addition, efflux of proline from preloaded cells preceded the increase in initial rates of proline entry. Taken together, we concluded that: 1) A system transport in primary cultures of this mammary adenocarcinoma is regulated by cell density as well as by availability of A system substrates, but these two types of regulation are kinetically distinct; and 2) starvation-induced enhancement of proline transport appears to be due to release from transinhibition, but may also involve a derepression-repression type of mechanism.

Amino Acids↗