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Biomedical subjects

L C Murphy

Publications and source records attributed to L C Murphy.

At least 91 records · Page 5Linked to original sources
PubMed1989

Endogenous growth factor expression in T-47D, human breast cancer cells, associated with reduced sensitivity to antiproliferative effects of progestins and antiestrogens.

To determine the functional significance of the endogenous expression of the epidermal growth factor (EGF) and transforming growth factor-alpha genes in T-47D, human breast cancer cells, we have examined the effects of two types of antiproliferative agents, progestins and antiestrogens, on the expression of these growth factors and the effects of exogenous EGF on the antiproliferative action of these agents. Using Northern blot analysis, the regulation of expression of these two genes by the antiproliferative agents, tamoxifen and monohydroxytamoxifen, was examined. In T-47D cells the two antiestrogens did not affect the accumulation of EGF mRNA and decreased the accumulation of TGF alpha mRNA. As we have shown before, the progestin, medroxyprogesterone acetate, increased the level of both EGF mRNA and TGF alpha mRNA in this cell line. The regulation of expression of these endogenous growth factor genes was unrelated to the proliferative behavior of T-47D cells since both antiestrogens and progestins were antiproliferative under the conditions of the experiments. The variant cell line, T-47D-5, had no detectable EGF mRNA and contained about 1/10th the level of TGF alpha mRNA expressed by "wild type" T-47D cells. T-47D-5 cells were 2.5 times more sensitive to the antiproliferative effects of both progestins and antiestrogens when compared to the growth factor expressing T-47D cells. Exogenously added murine EGF was able to decrease slightly the sensitivity of both cell lines to the antiproliferative effects of both progestins and antiestrogens as well as increase the proliferation of T-47D but not T-47D-5 cells. These data suggest that endogenous expression of growth factors may be associated with decreased sensitivity of the cells to growth inhibitory agents.

Breast Neoplasms↗
PubMed1989

The knee-chest position does not improve the efficacy of ipecac-induced emesis.

Previous studies have shown that ipecac-induced emesis, even if instituted very early, removes only a mean of 28% to 45% of an ingested tracer. Because vomiting is an ancient reflex that occurs in mammals, reptiles, and other animals, we speculated that, in humans, maintaining a sitting rather than a horizontal posture during induced emesis might decrease the efficacy of gastric emptying. To test this hypothesis, 20 normal fasting adult subjects underwent induced emesis in the knee-chest position on one day and in the sitting position on another. Twenty-five 100-micrograms tablets of cyanocobalamin were ingested as a tracer along with 250 mL tap water. Ten minutes after tracer ingestion, 30 mL ipecac syrup and 640 mL tap water were swallowed. All resulting vomitus was homogenized, frozen, and later assayed for cobalt using atomic absorption spectrophotometry. There was no difference in mean tracer recovery with the two positions: knee-chest, 47.2% v sitting, 46.9% (paired t test, P greater than .95). Analysis of cobalt recovery for all 40 episodes of emesis revealed a mean of 51.2 +/- 23.7 (SD) micrograms out of 108.7 micrograms total cobalt ingested (95% Cl, 43.6 to 58.7 micrograms). This represented 47.1% of the administered tracer dose (95% Cl, 40.1% to 54.0%). Even if initiated only ten minutes after an ingestion, ipecac-induced emesis removes an average of less than half of an ingested tracer dose, with a high degree of intersubject variability. Horizontal patient positioning does not appear to improve the efficacy of this procedure.

Adult↗
PubMed1989

Measurement of the relative binding affinity of zearalenone, alpha-zearalenol and beta-zearalenol for uterine and oviduct estrogen receptors in swine, rats and chickens: an indicator of estrogenic potencies.

1. The relative binding affinity of zearalenone, alpha-zearalenol, and beta-zearalenol for estrogen receptors was determined in the pig, rat and chicken. 2. Similar relative binding patterns were observed, with alpha-zearalenol exhibiting greater affinity than zearalenone and beta-zearalenol the least binding affinity in all species. 3. The relative binding affinity of alpha-zearalenol was greater in pig, than in rat and significantly greater than in chicken. 4. Interspecies differences in zearalenone sensitivity may be due to the binding affinity of alpha-zearalenol for estrogen receptors and differences in zearalenone metabolites formed.

Animals↗
PubMed1989

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells.

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1989

Variant estrogen receptor mRNA species detected in human breast cancer biopsy samples.

Thirty to 40% of estrogen receptor (ER) positive human breast tumors are resistant to endocrine therapy. To investigate the possibility that abnormal ER proteins may be associated with this endocrine resistant phenotype we have looked for the presence of abnormally sized ER mRNA in human breast tumor biopsies. Poly(A+) enriched RNA was isolated from 46 human breast tumor biopsies and analyzed by Northern blotting and hybridization with the human estrogen receptor OR-8 cDNA. Seventy percent of the tumors contained detectable 6.5 kilobase (kb) ER mRNA. Some tumors also contained smaller sized ER mRNA of approximately 3.8 and 2.4 kb. These variant sized transcripts were only detected in biopsies where the normal 6.5 kb mRNA was present. In some cases the abundance of the variant mRNAs was equal to or greater than the normal ER mRNA. The variant ER mRNAs were not due to nonspecific RNA degradation nor was there any evidence of gross alteration of the ER gene in tumors where variant mRNAs were detected. ER cDNA fragments representing only the E/F domain of the receptor showed little or no hybridization with the variant sized ER mRNAs, while a ER cDNA fragment covering the A/B, C and D domains hybridized to both the variant and normal ER mRNAs. This suggested that the smaller variant ER mRNAs may be missing some or all of the E/F domain which is thought to contain the steroid hormone binding domain of the receptor. It is hypothesized that if these intermediates were translated into viable proteins they may interfere with the normal ER function.

Biopsy↗
PubMed1989

The role of dipsticks in the diagnosis of urinary tract infection.

Dipsticks containing indicators for the presence of leucocytes (nephur test) and bacteria (nitrate test) were performed on 1134 urine samples obtained from children attending a Urinary Tract Infection Clinic. These dipstick results were compared with the results of standard leucocyte counts and culture of the same urines. The nitrite test was disappointing in diagnosing infection as it was positive in only 81.4% of urines with positive cultures. However, if it was combined with a nephur test only 3.9% of infections were missed. Conversely a positive nitrite test indicated with 91.3% accuracy that the urine was infected. Combined negative nephur and nitrite tests were 98.6% accurate in excluding urinary tract infection.

Evaluation Studies as Topic↗
PubMed1988

Epidermal growth factor gene expression in human breast cancer cells: regulation of expression by progestins.

Epidermal growth factor (EGF) is thought to be important in normal mammary development. The presence of EGF receptors in breast cancer cells suggests that it may also have a role in regulating growth of tumors of the human breast. Using a complementary DNA probe for the human EGF precursor we have examined expression of this gene in a series of human breast cancer cells in long term culture. The T-47D cell line demonstrated the highest level of EGF mRNA. EGF expression was not detectable in the MCF-7, BT 20, or HBL 100 cell lines. Surprisingly, in both T-47D and ZR 75 cells, pretreatment with progestins which exert antiproliferative effects under the conditions used increased EGF mRNA levels approximately 6-fold above untreated controls. This effect, demonstrable with as little as 0.1 nM of medroxyprogesterone acetate, was apparent as early as 12 h after addition of progestin and was reversed with the antiprogestin RU 486. Dexamethasone, estradiol, and dihydrotestosterone had no effect on EGF expression in T-47D cells. There was no evidence that the increased levels of EGF mRNA were due to gene amplification. Immunoprecipitation of biosynthetically labeled T-47D conditioned medium with antibodies to human EGF and EGF-precursor revealed the presence of both Mr 40,000 and 18,000 products. Fully processed Mr 6,000 EGF was not detectable in either conditioned medium or cell lysate. These data provide unequivocal evidence for the expression of the EGF gene in some human breast cell lines.

Breast Neoplasms↗
PubMed1988

Cloning and characterization of a cDNA encoding a highly conserved, putative calcium binding protein, identified by an anti-prolactin receptor antiserum.

Using antisera raised against partially purified rabbit mammary gland prolactin receptor, we have isolated a cDNA from a T-47D human breast cancer cell expression library which encodes the putative calcium-binding protein, calcyclin. Since the protein encoded by this cDNA co-purified with the prolactin receptor, we have tentatively named it a prolactin receptor-associated protein (PRA). Hybrid selection and in vitro translation showed that the protein encoded by this cDNA was approximately 10 kDa, a result confirmed by the amino acid sequence derived after DNA sequencing analysis. The PRA gene product has significant sequence similarity to the S-100 proteins, the cystic fibrosis antigen, and p11 proteins. The PRA/calcyclin cDNA obtained from the T-47D human breast cancer cell library has 37 nucleotides more 5'-untranslated information than that of the calcyclin cDNA from human fibroblasts. Since the transcription start site for the calcyclin gene has been confirmed in fibroblasts, our data suggest that a different transcription start site may be used in the T-47D cells. Isolation and nucleotide sequencing of the rat PRA cDNA showed there was 96% predicted amino acid sequence similarity with the human PRA and confirmed the highly conserved nature of the PRA/calcyclin gene between species. The PRA is expressed in most but not all human breast cancer cell lines, e.g. MCF 7. Northern analyses of RNAs from rat tissue indicated that PRA mRNA levels vary widely. They are low in the liver, brain, testes, and ovary, moderate in skeletal muscle, and high in the lung, kidney, and uterus. The availability of prolactin receptor-positive human breast cancer cell lines, which do (T-47D) and do not (MCF 7) express the PRA/calcyclin gene, will allow the investigation of the role, if any, of PRA in prolactin action.

Amino Acid Sequence↗
PubMed1988

Progestin regulation of EGF-receptor mRNA accumulation in T-47D human breast cancer cells.

Incubation of T-47D human breast cancer cells with the synthetic progestin, medroxyprogesterone acetate (MPA), resulted in a time and dose-dependent increase in epidermal growth factor-receptor (EGF-R) mRNA. Concentrations of MPA as low as 1 nM resulted in a greater than five fold increase in EGF-R mRNA. A significant increase (2-3 fold) in EGF-R mRNA was apparent 12 hr after exposure to MPA and a further increase was seen 12-48 hr after addition of MPA to the cultures. From these studies it is concluded that the increased EGF binding in progestin-treated T-47D cells results at least in part from increased EGF-R gene expression. We believe this is the first report of a steroid hormone modulating expression of this growth factor receptor gene.

Antineoplastic Agents↗
PubMed1987

Isolation and sequencing of a cDNA clone for a prolactin-inducible protein (PIP). Regulation of PIP gene expression in the human breast cancer cell line, T-47D.

We have initiated an analysis of the action of prolactin in a human breast cancer cell line (T-47D) by isolating and sequencing cDNA clones for a prolactin-inducible protein (PIP). PIP cDNA (approximately 600 base pairs) was isolated from a lambda gt11 expression library prepared from mRNA extracted from T-47D cells treated with prolactin and hydrocortisone. The identity of PIP cDNA was confirmed by hybrid-selected translation. PIP mRNA is a single mRNA species of approximately 900 bases which responds to lactogenic hormones (human prolactin and growth hormone) in a time- and dose-dependent manner. Treatment of T-47D cells with human growth hormone (hGH) increased PIP mRNA levels by 4.6-fold, while the combination of hGH and hydrocortisone or dihydrotestosterone had a more dramatic, 12.4-fold, effect. The latter steroid was the most potent. Dihydrotestosterone plus hGH increased transcription of the PIP gene by 3.7-fold. Dihydrotestosterone alone was as effective as dihydrotestosterone plus hGH in increasing the transcription rate, while hGH alone had no effect. Thus, lactogen may regulate PIP gene expression at a post-transcriptional step. PIP mRNA was expressed at low levels in other human breast cancer cell lines which were prolactin receptor-positive; MCF-7 and EFM-19 lines were exceptions. PIP cDNA had an open reading frame of 146 amino acids, sufficient to encode the precursor form of the secreted PIP protein (approximately 14.5 kDa). The similarity of the predicted amino acid sequence of PIP to the sequence of a protein encoded by a gene expressed in the mouse submaxillary gland prompted us to look for PIP in human saliva. Western blot analysis confirmed the presence of PIP in human saliva. Therefore, the PIP gene is useful in studying the molecular actions of the prolactin/growth hormone polypeptide hormone family and the interaction with androgen, in mammary and other potential target cells.

Amino Acid Sequence↗
PubMed1987

Expression of the gene encoding a prolactin-inducible protein by human breast cancers in vivo: correlation with steroid receptor status.

We have previously reported the identification and characterization of a prolactin-inducible protein (PIP) as well as the cloning of the gene encoding PIP in cultured human breast cancer cells. We now present three lines of evidence that the gene encoding PIP is also expressed by some human breast cancers in vivo: detection of PIP immunoreactivity in the serum of some breast cancer patients; immunohistochemical detection of PIP in breast cancer sections; and the presence of PIP mRNA, detected by complementary DNA hybridization in human breast biopsy samples. In a preliminary study using Western blot analysis authentic PIP was detected in the serum of some patients with breast cancer. Subsequently the sera of 234 unselected patients with breast cancer were assayed for the presence of PIP using a specific radioimmunoassay. Thirty-five % of these sera contained detectable PIP (i.e., greater than 3 ng/ml). As well as were able to show by immunohistochemical techniques that PIP immunoreactivity was present in some human breast biopsy specimens. Levels of estrogen receptor, progesterone receptor, and PIP mRNA were determined in an unselected population of 51 human breast tumor biopsies. Sixty-one % of these tumors had detectable PIP mRNA; a positive correlation (r = 0.52; P less than 0.01) was found between PIP mRNA levels in breast biopsy samples and estrogen receptor content, a known prognostic indicator in human breast cancer.

Biopsy↗
PubMed1987

Estrogen induction of N-myc and c-myc proto-oncogene expression in the rat uterus.

The mechanisms involved in the proliferative response of the uterus to estrogen are poorly understood. The c-myc proto-oncogene has recently been shown to be rapidly activated in quiescent cells exposed to various mitogens. We have examined expression of c-myc and a closely related proto-oncogene, N-myc, in the rat uterus after in vivo administration of 17beta-estradiol (E2), 5 micrograms/100 g body weight, to prepubertal ovariectomized rats. Maximal c-myc messenger RNA (mRNA) accumulation, as determined by densitometric analysis of Northern blots of poly (A)+ uterine RNA was observed 3 h after E2 treatment. Maximal expression of c-myc was 8.6 +/- 0.8-fold (mean +/- SEM for 3 separate experiments) compared to basal levels seen in vehicle-treated ovariectomized rats. The maximal level of c-myc mRNA in the E2-stimulated uterus was higher (3- to 6-fold) than that observed in uteri from intact rats in either diestrous or the proestrous-estrous stages of the estrous cycle. There was no significant difference in the level of uterine c-myc mRNA throughout the estrous cycle. Under stringent conditions, the N-myc DNA probe hybridized with a single 3 kilobase (kb) transcript which was virtually undetectable in ovariectomized rat uteri and increased 6-fold within 15 min after E2 treatment. Maximal induction was seen 30-60 min post E2 treatment. At 1 h post E2 the level of N-myc mRNA was 9.3 +/- 0.4-fold (n = 3) compared to vehicle-treated rats. Under conditions of slightly reduced stringency, N-myc DNA also hybridized with a 2.2 kilobase transcript. Expression of the N-myc related gene also occurred more rapidly after E2 administration than c-myc mRNA. Our in vivo data are analogous to the in vitro observations that mitogen stimulation of quiescent cells results in a rapid accumulation of myc proto-oncogene mRNAs. In cycling cells in vitro and in the uterus of intact rats throughout the estrous cycle, the level of expression of the myc oncogenes is relatively constant. Since expression of the c-myc and N-myc proto-oncogenes appears to be restricted to different cell and tissue types our data indicate that there is at least one cell type present in the quiescent uterus that is able to respond rapidly to E2. The rapidity of the N-myc response would argue for a direct effect of E2. In contrast the c-myc response is considerably delayed and may be mediated via autocrine, paracrine, or circulating estrogen-dependent growth factors.

Actins↗
PubMed1987

Estrogen induces insulin-like growth factor-I expression in the rat uterus.

The inability to convincingly demonstrate a mitogenic effect of estrogen on isolated uterine cells in culture suggests that autocrine or paracrine growth factors may be important in the estrogen-induced uterine proliferative response. Here we report that uterine expression of insulin-like growth factor-I (IGF-I), an important mediator of GH action, is increased after 17 beta-estradiol (5 micrograms/100 g bw, ip) administration to ovariectomized prepubertal rats. An increase in uterine IGF-I mRNA abundance, approximately 14-fold above untreated controls, was apparent 6 h after estrogen administration and the level achieved exceeded that seen in the uterus from intact mature rats during diestrus. In contrast to the increase in IGF-I expression in the uterus, no significant change in serum IGF-I concentration or hepatic or renal IGF-I mRNA abundance was demonstrable after 17 beta-estradiol injection of ovariectomized prepubertal rats. The increase in uterine IGF-I expression, was similar in both pituitary-intact and hypophysectomized, ovariectomized rats. We believe this is the first report of induction of IGF-I expression by estrogen in vivo. As such, the finding expands the role and significance of IGF-I as a mediator of growth beyond that related to GH.

Animals↗
PubMed1986

Modulation of lactogenic receptors by progestins in cultured human breast cancer cells.

Progesterone receptors (PgR) are present in many breast cancers, but few specific actions of progestins in breast cancer cells have been reported. We now report that progestins specifically modulate lactogenic receptor expression in cultured T-47D and MCF-7 human mammary carcinoma cells. When T-47D cells were preincubated for 24 h with 1 nM medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methyl-4-pregnene-3,20-dione), specific binding of [125I]human GH ([125I]hGH) and [125I]human PRL was increased to 205 +/- 22% (+/- SE) (P less than 0.01) and 175 +/- 32% (P less than 0.05), respectively, of that in control cultures. There was no significant effect on cell number and no significant enhancement of specific binding of [125I]porcine insulin, [125I]salmon calcitonin, [125I]human transferrin, or [3H] Concanavalin A. Lactogenic receptor number was increased from 6,490 +/- 500 (n = 12) to 13,180 +/- 3,270 (n = 7; P less than 0.01) sites/cell, with no significant change in affinity for hGH. Progesterone, which is readily metabolized by these cells, was less potent than the synthetic progestins (medroxyprogesterone acetate, R 5020 (17 alpha, 21-dimethyl-19-norpregn-4,9-diene-3,20-dione), and ORG 2058 (16 alpha-ethyl-21-hydroxy-19-norpregn-4-en-3,20-dione), but physiological concentrations of progesterone (1 nM) significantly enhanced specific binding of [125I]hGH to 153 +/- 23% of the control value (n = 6; P less than 0.05). Physiological concentrations of androgens, estrogens, and glucocorticoids had no significant effect. MCF-7 cells were considerably less sensitive to these effects of progestins than T-47D cells, probably due to the lower PgR concentration in MCF-7 cells. These observations, which indicate that lactogenic receptor expression is controlled, at least in part, by progestins in these mammary carcinoma cell lines, may have important implications in the management of human breast cancer, where high levels of this receptor may reflect a functional PgR and a highly hormone-dependent phenotype.

Breast Neoplasms↗