PubMed Health⌕ Search

Biomedical subjects

M E Lippman

Publications and source records attributed to M E Lippman.

At least 253 records · Page 14Linked to original sources
PubMed1984

Isolation and characterization of an estrogen-inhibited variant derived from the MCF-7 breast cancer cell line.

The mechanism by which pharmacological concentrations of estrogen can paradoxically inhibit the growth of human breast cancer is unknown. We have selected for a variant line of MCF-7 which may help to understand this process. The variant was selected by exposing MCF-7 cells to high-specific-activity 16 alpha-[125I]iodoestradiol. These cells were viably frozen for two isotopic half-lives, defrosted once, then reexposed to 16 alpha-[125I]iodoestradiol to allow maximal radiation damage mediated by isotope associated with binding sites. This cell line (113) is one of 55 lines cloned from MCF-7 cells that survived this treatment. The growth response to estradiol of the 113 breast cancer cells grown in monolayer is normal for 4 to 6 days, and then the cell number plateaus as the cells appear to round up and detach. Concomitantly, a decrease in [3H]thymidine incorporation occurs. The cells cannot be rescued by removing estradiol from the medium. The inhibition is dose dependent and can be seen in concentrations of estradiol as low as 10(-10) M. The 113 cells are also inhibited by antiestrogens. They have normal levels of estrogen receptors which bind to DNA cellulose with activation. Progesterone receptors are estrogen inducible, although the levels are one-third that of wild-type MCF-7 cells. The morphological changes determined by electron microscopy of estrogen-treated cells are typical of degenerative cells. To investigate the possibility that inhibitory factors are secreted into the medium by 113 cells, conditioned medium from estrogen-exposed 113 cells is added to normal MCF-7 and 113 cells. No decrease in [3H]thymidine incorporation compared to controls is observed. When the secreted proteins are labeled with [35S]methionine and analyzed by sodium dodecyl sulfate-acrylamide gels, no major differences are apparent in the 113 and MCF-7 cells. Thus, the source of the defect is still unknown. It remains to be seen if the growth-inhibitory effects of 17 beta-estradiol on this cell line are receptor mediated or related to specific gene products which can be identified.

Breast Neoplasms↗
PubMed1983

Continuous estrogen exposure in the rat does not induce loss of uterine estrogen receptor.

Cytosol estrogen receptor (ERc) and nuclear estrogen receptor (ERN) levels were investigated in rat uteri under different conditions of hormonal exposure. The amount of directly assayable receptor was closely related to the serum concentration of 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2). A double injection technique was established to maintain serum levels of [3H]E2 which were sufficient to saturate receptor sites. Under these conditions, stable ERC and ERN levels are maintained throughout the study period. 30% of the total ER remains cytoplasmic in localization despite continuous hormonal exposure. Properties of ERC and ERN after 6 h of continuous hormonal exposure were investigated and found to be different from receptors found in these subcellular compartments 30 min after hormone injections. ERC from uteri 30 min after injection showed a faster sedimentation coefficient than ERC prepared 6 h after hormone treatment. ERC after 6 h of hormonal exposure showed a reduction of binding to calf thymus DNA adsorbed on cellulose in a cell-free system. ERC 30 min after [3H]E2 treatment had a biphasic dissociation pattern consistent with two different receptor populations, whereas uterine ERC obtained after 6 h of in vivo exposure to estradiol showed virtually no dissociation at 22 and 28 degrees C. In contrast to ERC, ERN 6 h after hormone injection sedimented faster than ERN obtained 30 min after treatment. KCl extractable ERN obtained either at 30 min or 6 h posthormone treatment showed biphasic dissociation kinetics at 22 and 28 degrees C, whereas KCl nonextractable ERN showed virtually no dissociation. Virtually all of the specifically bound ligand in cytosol and nuclear preparations was proven to be authentic E2. We conclude that total cellular receptor is quantitatively conserved during 6 h of continuous hormonal treatment. Nuclear receptor loss is not a requisite for receptor-mediated steroid function, although important time-dependent changes in receptor properties in both cytoplasmic and nuclear compartments do occur.

Animals↗
PubMed1983

Characteristics of different cytoplasmic and nuclear estrogen receptors appearing with continuous hormonal exposure.

Biochemical properties of cytosol estrogen receptor (ERC) and nuclear estrogen receptor (ERN) from rat uteri continuously exposed in vivo to 17 beta-[2,4,6,7-3H] estradiol ( [3H]E2) for 6 h have been studied on the basis of immunological recognition and chromatographic elution patterns. Overall concentrations of ERC and ERN did not change during this time period when receptor-saturating concentrations of [3H]E2 were maintained (Jakesz, R., Kasid, A., and Lippman, M. E. (1983) J. Biol. Chem. 258, 11798-11806); however, biochemical characteristics were different in ERC and ERN after short or long term hormonal exposure. When ERC from rats treated with estradiol for 30 min was applied to HAP or DEAE columns, two different ER binding components were seen. DNA binding in a cell-free system revealed that these binding components represented an activated and a nonactivated ERC population. After long term hormonal exposure (6 h), only one component of ERC with low DNA binding could be shown despite the preservation of an equivalent quantity of cytoplasmic binding activity. This binder does not react with a monoclonal antibody directed against extranuclear estrogen receptor species. These data suggest disappearance of the activated ERC population, with appearance of a new, immunologically nonrecognizable ERC species with 6 h of continuous hormonal exposure. Elution profiles of ERN on HAP chromatography reveal 2 different binding components at 30 min and at 6 h of continuous [3H]E2 exposure. There is an increase of the population eluted at higher molarity after 6 h of in vivo treatment. This later eluting binding component is the major DNA binder in vitro. ERN from both time points are recognized immunologically by monoclonal antibody. After reaction with the antibody, the sedimentation coefficient shifted to 8-9 S on sucrose gradients, but the previously described faster sedimentation of ERN extracted 6 h after injection persisted. We conclude that ER in both cellular compartments undergoes time-dependent alterations, which may be involved in the initiation of hormone action.

Animals↗
PubMed1983

Efforts to combine endocrine and chemotherapy in the management of breast cancer: do two and two equal three?

Standard approaches to the systemic management of breast cancer have probably reached a plateau. Endocrine therapy leads to responses in only 1/3 of patients, probably even less if no clinical preselection is applied, and the median benefit probably does not exceed one year. Although three-drug chemotherapeutic combinations yield responses in 55-65% of patients, the median duration of response is at best only 10 months, and addition of more drugs does not seem to offer much additional benefit. Although combining these approaches seems attractive initially, the theoretical improvement would be modest even if the effects were additive, and in fact the endocrine component may well perturb the cell cycle kinetics to reduce sensitivity to the cytotoxic component so that results would be less than additive. Clinical trials of combined chemo-endocrine regimens have tended to evaluate only the initial response rate, whereas a valid comparison must consider the total response duration and survival given either combined or the usual sequential approaches. Combining endocrine and cytotoxic chemotherapies may have a greater effect when a more rational means for their amalgamation is employed. Early trials using endocrine manipulation to synchronize cell sensitivity to cytotoxic treatment have been promising. Increasing attention needs to be directed towards more innovative trial design which is respectful of the biological realities of cancer.

Adrenalectomy↗
PubMed1983

Characterization of insulin regulation of lipid synthesis in MCF-7 human breast cancer cells.

Stimulation of lipid synthesis by insulin in MCF-7 human breast cancer cells is characterized by an increase in acetate incorporation into long-chain fatty acids. The effects occurs in the absence of an increase in glucose uptake by the cells, and cannot be explained by a decrease in turnover of cellular fatty acids. Differential substrate experiments as well as direct measurement of enzyme activities indicate that insulin stimulates increases in activity of the first enzyme of the de novo pathway, acetyl CoA carboxylase. [32Pi] incorporation into phospholipids is also stimulated by insulin. Thin layer chromatography reveals five peaks of [32Pi]-labeled phospholipids corresponding in mobility to the following standards: lysophosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. [32Pi] incorporation into each of these peaks is stimulated, although the degree of stimulation varies.

Breast Neoplasms↗
PubMed1983

Psychosocial and physical outcomes of primary breast cancer therapy: mastectomy vs excisional biopsy and irradiation.

Thirty-eight patients treated for primary breast cancer as part of a prospective randomized clinical trial were questioned retrospectively as to their psychosocial adaptation to treatment. Twenty patients had received mastectomy and eighteen had received excisional biopsy plus radiation of the intact breast. Aside from body image concerns, there were no marked psychosocial differences detected between these groups. Previous studies emphasizing serious psychological problems in mastectomy patients and fewer such problems in nonmastectomy patients may be influenced by biases that are not present in a randomized study design.

Adult↗
PubMed1983

Protein-associated intercalator-induced DNA scission is enhanced by estrogen stimulation in human breast cancer cells.

Estrogen-responsive human breast cancer cells (MCF-7) displayed a higher frequency of intercalator-induced protein-associated DNA scission after treatment with 17 beta-estradiol (E2) than did cells that had not received estrogen treatment. This effect was dependent on estrogen concentration (maximum enhancement at approximately equal to 1 nM E2) and time (maximum effect seen approximately equal to 24 hr after E2 addition). Human breast cancer cells lacking estrogen receptors did not display the enhanced response. Antiestrogens produced a slight decrease in intercalator-induced DNA scission, whereas insulin produced an enhanced effect. The DNA breaks produced by the intercalators 5-iminodaunorubicin and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in these cells were undetectable without enzymatic deproteinization of cell lysates prior to quantification by alkaline elution. Intercalator-induced DNA-protein crosslinking also was enhanced in E2-treated MCF-7 cells. Studies with m-[14C]AMSA revealed no estrogen-associated increases in drug uptake. The data suggest that E2 treatment, either by specifically and directly increasing active transcription in chromatin or through secondary effects on DNA that accompany alterations in cell growth or cell cycle distribution, alters the susceptibility of DNA to intercalator-induced protein-associated DNA scission. If this enhanced protein-associated scission is selectively localized to transcriptionally active chromatin, the adsorption of the DNA-bound proteins to membrane filters (DNA-protein crosslinking) may allow identification and isolation of estrogen-regulated gene sequences.

Breast Neoplasms↗
PubMed1983

Androgen receptor characteristics in skin fibroblasts from men with pubertal macromastia.

Endocrine studies in men with pubertal macromastia (PM) have failed to reveal a hormonal abnormality to account for the disorder. As a result, it has been hypothesized that this form of gynecomastia may be a manifestation of a target organ abnormality. Because the syndromes of apparent androgen resistance, such as testicular feminization and Reifenstein's Syndrome, are associated with gynaecomastia, we examined skin fibroblasts cultured from men with pubertal macromastia for androgen receptor defects. We studied 12 men with PM and confirmed that plasma concentrations of testosterone, oestradiol, gonadotrophins, and prolactin, were all within the normal range; findings identical to those of a similar series of patients previously reported. Androgen receptor content (R0) and binding affinity (Kd) in cultured areolar and pubic skin fibroblasts were measured using a dispersed, whole cell assay. Nineteen areolar cell lines from the 12 patients with PM were compared with 4 areolar cell lines from three normal men and 9 areolar cell lines from nine normal women. There was no difference in the mean androgen receptor content (approximately 10 000 sites/cell) or binding affinity (approximately 1 nM) between the patients' fibroblasts and those of the normal subjects. Similarly, there were no differences in these parameters when pubic skin fibroblast androgen receptors were used for the comparison. We conclude that, although PM may yet be due to a defect in breast tissue sensitivity to androgen, the disorder cannot be explained by abnormalities in fibroblast androgen receptor number or affinity.

Adolescent↗
PubMed1983

Melatonin-induced increase in cytoplasmic estrogen receptor activity in hamster uteri.

The pineal gland hormone, melatonin, has been shown to influence the growth and metabolism of uterine tissue in the Syrian hamster. Because the uterus is an estrogen-responsive target tissue, we studied the effect of melatonin on the unoccupied estrogen receptor (ER) activity of immature hamster uteri in vivo and in vitro. A single sc injection of 2.5 micrograms melatonin increased the ER activity of uterine cytosol (Rc) 30% within 60 min [from 147.4 +/- 14.1 (SE), to 191.8 +/- 18.5 fmol/mg protein]. Scatchard analysis showed the induced population of receptor to be homogeneous and of high affinity, with an equilibrium dissociation constant (Kd = 0.08 nM) similar to that for uteri for vehicle-treated animals. The induction of Rc by melatonin persisted for 90 min, but Rc returned to control levels by 2 h after injection. Scatchard analysis of unoccupied nuclear receptor showed no significant differences either in the quantity of receptor (vehicle = 379.7 +/- 46.7, melatonin = 396.9 +/- 76.7 fmol/mg protein) or in the Kd (vehicle = 0.75 nM, melatonin = 0.60 nM) between these two groups. In vitro incubation of fresh whole uteri for 60 min in media containing 10(-5) M melatonin increased Rc by 83% (from 41.3 +/- 10.7 to 75.4 +/- 11.0 fmol/mg protein), supporting a direct effect of melatonin on uterine tissue. The Rc induced by melatonin both in vivo and in vitro was sensitive to the degree of homogenization used in preparation of the cytosol. Whereas conservative homogenization (two 10-sec bursts with an intermittent 40-sec cooling period) of uteri from melatonin-treated animals was associated with a significant increase in Rc, homogenization for 60 sec without intermittent cooling showed a significant decrease (from 251.1 +/- 17.4 to 182.7 +/- 5.8 fmol/mg protein) as compared with uteri from vehicle-treated animals. Whole organ analysis of uteri from animals injected with melatonin followed by [3H]estradiol, in which no homogenization was used, demonstrated a 26% increase in Rc specific binding, confirming induction as the primary response to melatonin. These findings demonstrate the ability of melatonin to modulate ER activity in hamster uteri, and may help explain the effects of melatonin on uterine growth and metabolism.

Animals↗
PubMed1983

Antiestrogenic effects of LY 117018 in MCF-7 cells.

LY 117018 is a nonsteroidal compound which has been shown to antagonize more effectively than tamoxifen the effects of estradiol in rats and mice. We have further defined the properties of this compound on cell growth and progesterone receptor (PR) induction and have compared inhibitory effects to relative binding affinity in a human breast cancer cell line. In MCF-7 cells, LY 117018 [( 6-hydroxy-2-(p-hydroxyphenyl)-benzo(b) thien-3-yl-p-(2-(pyrrolidinyl)ethoxy phenyl ketone] is devoid of estrogenic effects, has a greater affinity for the estrogen receptor (ER) than tamoxifen at 4 C, and is 100-1000 times more potent than tamoxifen at inhibiting cell growth. While tamoxifen has been reported to induce PR, a purported estrogen effect, the LY 117018 compound failed to induce PR using a broad range of concentrations. The direct antagonistic effect of LY 117018 was demonstrated by the use of varying concentrations of LY 117018 and estradiol concomitantly. We conclude that LY 117018 is a very effective antiestrogen devoid of any stimulatory effects among these parameters tested.

Binding, Competitive↗
PubMed1983

Evaluation of tamoxifen doses with and without fluoxymesterone in advanced breast cancer.

From a group of 108 female patients with measurable and/or evaluable metastatic breast carcinoma, 52 were randomized to receive tamoxifen and 56 to receive tamoxifen and fluoxymesterone. The fluoxymesterone dose, given orally twice a day, was 7 mg/m2 body surface area. The tamoxifen dose per patient, also given orally twice a day, ranged from 2 to 100 mg/m2 body surface area. Eighty-five percent of the patients had received previous chemotherapy and 60% previous hormone therapy. The complete and partial remission rate was better with the tamoxifen and fluoxymesterone regimen (p = 0.016), with remission rates of 15% for tamoxifen alone and 38% for the combination. The tamoxifen and fluoxymesterone regimen appeared to have higher remission rates in all subsets of pretreatment variables. Duration of remission with each regimen was similar, but the overall time to treatment failure for tamoxifen and fluoxymesterone was longer than for tamoxifen alone (180 versus 64 days, p = 0.01). Median survival with the combination was 380 days compared to 330 days for tamoxifen. No significant dose-response relationships emerged. Side effects were not different between dose levels or regimens except for the androgen effects in the tamoxifen and fluoxymesterone combination. These results suggest that there is no major dose-response effect for doses ranging from 2 to 100 mg/m2 body surface area given twice daily in this largely (94%) postmenopausal pretreated patient group, and that the tamoxifen and fluoxymesterone regimen is superior to tamoxifen alone.

Adult↗
PubMed1983

Antiestrogen therapy of breast cancer.

Studies in vitro and in animal models have helped define the mechanism of action of antiestrogens and the differences in the physical properties of the various antiestrogens. Tamoxifen is the most widely studied antiestrogen. A review of 45 separate studies has shown a response rate to tamoxifen in advanced breast cancer that varies between 14% and 57%, with an overall response rate of 34%; prior therapy strongly influenced response rate. Menopausal status did not seem to influence response, and no dose-response relationship has been documented. Patients with estrogen-receptor-positive tumors are more likely to respond than those with estrogen-receptor-negative disease. Tamoxifen has been shown to be an unusually safe and well-tolerated drug. Studies of antiestrogens as adjuvants to local therapy of early breast cancer have generally produced encouraging results. More studies are needed, however, in order to define those subsets of patients who are most likely to benefit from adjuvant therapy with tamoxifen and to determine the most effective regimens.

Aged↗
PubMed1983

Comparison of dye exclusion assays with a clonogenic assay in the determination of drug-induced cytotoxicity.

The following factors must be considered when dye exclusion assays are interpreted. (a) It may require several days for lethally damaged cells to lose their membrane integrity following a cytotoxic insult. (b) During this time, the "surviving" cells may continue to proliferate. (c) Also during this time, some lethally damaged cells may undergo an early disintegration, so that they are not present to be stained with dye at the end of the culture period. Factors b and c may cause an underestimate of cell kill when the results of the assay are based upon the traditional "percent viability" expression. In order to overcome these problems, an internal standard was developed and tested. This was based upon the addition of a constant number of permanently fixed duck erythrocytes to the cultures of cells from two different established tumor cell lines. Results were based upon comparisons of the ratios of "viable" tumor cells to duck erythrocytes on permanent cytocentrifuge slides prepared from the cultures. This novel "ratio" method was found to be a more sensitive index of drug-induced cell kill than the traditional percent viability method. A standard agar cloning assay gave somewhat higher estimates of cell kill than the ratio method, although both assays were in qualitative agreement for the drugs tested. All three assays demonstrated a clear dose-effect relationship for most of the drugs tested. Dye exclusion assays may have a useful role in chemosensitivity testing in vitro.

Animals↗
PubMed1983

Hormonal regulation of de novo pyrimidine synthesis and utilization in human breast cancer cells in tissue culture.

The hormonal regulation of pyrimidine synthesis and utilization is examined in the estrogen-responsive MCF-7 human breast cancer cell line. [14C]Acetate is used as a novel marker in assaying the incorporation of pyrimidine nucleotides derived through de novo synthesis into DNA. Technical verification of the validity of this tracer in DNA synthesis is presented. The experimental conditions permitting extrapolation from radioactivity incorporated into DNA to pmol of DNA synthesized/unit of time are discussed. We observe that estrogen administration increases the incorporation of [14C]acetate into DNA, and this effect is most evident after 36 hr of exposure to hormone. Treatment with the antiestrogen tamoxifen produces substantial reductions in the rate of incorporation of [14C]acetate into DNA. Incorporation correlates well with net rates of DNA synthesis, as measured by 32Pi incorporation into DNA. Concomitant administration of thymidine in concentrations in excess of 10(-7)m partially relieves tamoxifen inhibition of net DNA synthesis and increases the apparent stimulation of net DNA synthesis following 18 to 32 hr of estrogen treatment. Activity of de novo enzymes such as carbamylphosphate synthetase, aspartate transcarbamylase, orotidine pyrophosphorylase, and orotidine decarboxylase also varies with hormone treatment. All characteristically increase following estrogen treatment and decrease after tamoxifen administration. These results suggest that changes in de novo pyrimidine synthesis after hormone treatment are directly linked to hormone-induced alterations in the activity of specific-enzymes in the de novo pathway.

Acetates↗
PubMed1982

Dihydrofolate reductase gene amplification and possible rearrangement in estrogen-responsive methotrexate-resistant human breast cancer cells.

Methotrexate-resistant (MTXR) human breast cancer cells have been obtained which are 1000-fold less sensitive to this drug than the wild type MCF-7 cells from which they were derived. The resistant cells contain approximately a 25-fold increase in the activity of the target enzyme dihydrofolate (DHF) reductase. Enzyme inhibition studies and methotrexate affinity studies fail to reveal any difference in the DHF reductase present in the MTXR cells compared to wild type MCF-7 cells. Cytogenetic analysis demonstrates the presence of elongated marker chromosomes in the resistant cells which are not found in the parental cell line. Analysis of the DNA from MTXR and wild type MCF-7 cells using Southern blot hybridization indicates that the MTXR MCF-7 cells contain more copies of DHF reductase gene sequences than do wild type MCF-7 cells. These experiments also suggest that the amplified DHF reductase gene sequences in MTXR cells may have undergone a uniform structural rearrangement involving the 5' flanking sequences during the process of amplification. MTXR MCF-7 cells respond to estradiol by increasing cell growth, and the level of DHF reductase in the MTXR cells is further induced following administration of estradiol. Radiolabeling studies demonstrate that estrogen stimulates the actual synthesis of DHF reductase in human breast cancer cells.

Breast Neoplasms↗
PubMed1982

Effects of RNA and ribonuclease on the binding of estrogen and glucocorticoid receptors from MCF-7 cells to DNA-cellulose.

Effects of RNA and ribonucleases on the binding of estrogen- and glucocorticoid-receptor complexes from MCF-7 cells to calf thymus DNA-cellulose have been studied under cell-free conditions. Exogenous ribonucleases can increase binding of estrogen- and glucocorticoid-receptor complexes to DNA-cellulose. Purified RNA derived from MCF-7 cells can inhibit estrogen- and glucocorticoid-receptor binding to DNA-cellulose. Under low salt conditions, RNA appears to be associated with estrogen-receptor complexes from MCF-7 cells. These observations taken together suggest that there is an interaction between steroid hormone-receptor complexes and RNA. The implications of this interaction are discussed.

Breast Neoplasms↗
PubMed1982

A new model system for studying the phosphatidylinositol cycle.

An early manifestation of the response of WRK-1 rat mammary tumor cells to vasopressin is an increase in incorporation of (32P)Pi into phospholipids. Incorporation into all classes of phospholipids is stimulated; however, incorporation into phosphatidylinositol (PI) is increased to the greatest degree (3- to 10-fold as compared with 1.3- to 2-fold for the other phospholipids). Furthermore, increased incorporation into PI is accompanied by an increased rate of PI turnover; turnover rates of the other phospholipids are unaffected by vasopressin.

Acetates↗